Inhibition of migration inhibitory factor in the treatment of diseases involving cytokine-mediated toxicity

ABSTRACT

The present invention relates to diagnostic methods for determining migration inhibitory factor (MIF) mRNA content in a sample, wherein MIF is human MIF polypeptide having a molecular weight of approximately 12.5 kDa and kits for detecting MIF.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of Ser. No. 11/225,124 filed Sep. 14,2005, now abandoned, which is a continuation of divisional of Ser. No.09/557,823, filed Apr. 25, 2000, now U.S. Pat. No. 6,998,238; which is adivisional of Ser. No. 08/471,586, filed Jun. 6, 1995, now U.S. Pat. No.6,080,407; which is a divisional of Ser. No. 08/462,350, filed Jun. 5,1995, now abandoned; which is a continuation-in-part of Ser. No.08/243,342, filed May 16, 1994, now abandoned; which is acontinuation-in-part of Ser. No. 08/063,399, filed May 17, 1993, nowabandoned. The entireties of the aforementioned applications areincorporated herein by reference.

1. INTRODUCTION

The present invention relates to compositions and methods for inhibitingthe release and/or biological activity of migration inhibitory factor(MIF). In particular, the invention relates to the uses of suchcompositions and methods for the treatment of various conditionsinvolving cytokine-mediated toxicity, which include, but are not limitedto shock, inflammation, graft versus host disease, and/or autoimmunediseases.

2. BACKGROUND OF THE INVENTION

2.1. Cytokines in Septic Shock

Septic shock is a multifaceted pathological condition characterized mostprominently by deleterious hemodynamic changes and coagulopathy leadingto multiple organ failure and often to death. The altered physiologicalmechanisms underlying the septic shock syndrome, and the cellular meansby which these changes are induced and controlled, are not yet known inprecise detail. In broad outline, however, a consensus view of eventsculminating in septic shock has emerged over the last several years. Inparticular, it is now generally accepted that septic shock reflects theindividual, combined and concerted effects of a large number ofendogenous, host-derived mediator molecules. These mediators areproduced in response to initiating stimuli that indicate the host hasbeen invaded, and the class of peptide mediators that were generallyfirst recognized as white blood cell products have come to be known ascytokines. As mediators of toxic effects and pathological alterations inhost homeostasis, these endogenous factors represent potentiallyattractive therapeutic targets, and septic shock remains a potentiallylethal cytokine-mediated clinical complication against which there is nogenerally effective therapeutic approach.

Although traditionally termed “septic” shock, infection by a variety ofmicroorganisms including not only bacteria but also viruses, fungi, andparasites can induce septic shock. In fact, the shock syndrome is moreproperly associated with the host's response to invasion rather thanjust infection, as cancer and trauma, for instance, can also serve asinitiators. In the case of infection by gram-negative bacteria, one ofthe best studied examples, it is believed that the appearance ofbacterial endotoxins such as lipopolysaccharide (LPS) in the hostbloodstream leads to the endogenous production of a variety of hostfactors that directly and indirectly mediate the toxicity of LPS, whichitself is relatively innocuous for most cells. These host-derivedmediators include many now well-recognized inflammatory cytokines andclassical endocrine hormones in addition to a number of other endogenousfactors such as leukotrienes and platelet activating factor. It isgenerally acknowledged, however, that the full cast of participants andeach of their interrelated roles in the host response remainsincompletely appreciated.

In general, those mediators that appear earlier in an invaded host arethought to trigger the release of later appearing factors. Also, manyendogenous mediators not only exert direct effector functions at theirtarget tissues, but also prime local and remote tissues for subsequentresponses to other mediators. This interacting network of host factorshas been termed the “cytokine cascade”. This term is meant to indicaterapid extension and amplification of the host response in such a waythat only one or a few initiating stimuli trigger the eventual releaseand participation of scores of host mediators. Although a number offeatures of the host response are thought to assist in fighting offinvasion, an overly robust or poorly modulated endogenous response canrapidly accelerate to rapidly produce such profound alterations in hosthomeostasis at the cellular, tissue, and systemic levels that death mayensue within hours.

Among the interacting factors that together comprise the cytokinecascade, the cytokine known as tumor necrosis factor-alpha (TNFα) is themost important identified to date. TNFα is the first cytokine to appearin the circulation after LPS challenge. The hemodynamic and metabolicalterations that result from the experimental administration of TNFαclosely resemble those that have been observed in endotoxemia and septicshock. In animal models, TNFα is the only host factor which itself caninitiate a lethal syndrome that mimics septic shock in detail. In thisrespect, TNFα can be considered a sufficient mediator of septic shock.Functionally neutralizing TNFα antagonists such as anti-TNFα antibodiesare protective in otherwise lethal bacterial infections, and in thisrespect TNFα can be considered a necessary mediator of septic shock.

Other cytokines participate in the host response to LPS but appear laterin the circulation. However, no other cytokine has been shown to be bothnecessary and sufficient to mediate septic shock. For example, certaininterleukins (IL-1, IL-6 and IL-8) which appear in serum more than 2hours after LPS challenge, and interferon γ (IFN-γ) which appears after6 hours, are thought to play a significant role in septic shock, and canbe shown to contribute to lethality in certain disease models or underexperimental conditions of endotoxemia. Antagonism of the effects ofspecific interleukins and interferons has been shown to confer asignificant protective effect under certain conditions. Nevertheless,none of these other factors can itself induce a full-blown septicshock-like effect in otherwise healthy individuals, and none of theseother cytokines appears to play as central and critical role in septicshock as TNFα.

In view of the foregoing, TNFα stands as an ideal target for thetreatment of septic shock. Unfortunately, temporal characteristics ofthe endogenous TNFα response suggest a significant practical limitationfor this potential therapy. TNFα, one of the earliest elicited mediatorsto appear in acute disease, rapidly peaks after bolus endotoxinchallenge (30-90 minutes), and diminishes just as promptly. It ispresumed that most of the damaging effects of TNFα in septic shock arecompleted during this early period, hence TNFα antagonists such asanti-TNFα antibodies would ideally be present at this time. Since thistherapeutic window is apparently so short and occurs so early, thetimely delivery of anti-TNFα based therapeutics may be very difficult toachieve clinically.

Therefore, in order utilize cytokines as targets for the treatment ofseptic shock and other cytokinemediated toxic reactions, there exists adesperate need to discover additional targets that are both necessarycomponents of the cytokine cascade and occur at a time during theendogenous response that is accessible for therapeutic antagonism in thecourse of clinical treatment.

2.2. The Pituitary as a Source of Protective Cytokines

Recent studies suggest that the pituitary gland may produce factors thatinhibit endotoxin-induced TNFα and IL-1 production and thus may serve asa source for potentially protective factors that may be used to treatshock and/or other inflammatory responses. (Suzuki et al., 1986, Am. 3.Physiol. 250: E470-E474i; Sternberg et al., 1989, Proc. Natl. Acad. Sci.USA 86: 2374-2378; Zuckerman et al., 1989, Eur. J. Immunol. 19: 301-305;Edwards III et al., 1991a, Endocrinol. 128: 989-996; Edwards III et al.,1991b, Proc. Natl. Acad. Sci. USA 88: 2274-2277, Silverstein et al.,1991, J. Exp. Med. 173:357-365). In, these studies, hypophysectomizedmice (i.e., animals that have had their pituitary glands surgicallyremoved) exhibited a marked increased sensitivity to LPS injectionrelative to sham-operated control mice. In fact, the LPS LD₁₀₀ forcontrol mice was approximately 1-2 logs higher than that determined forthe hypophysectomized mice, suggesting that the pituitary gland producesone or more factors that may act to increase the host animals ability toresist endotoxin challenge. Some of these studies implicate theinvolvement of ACTH and adrenocorticosteroids (e.g., Edwards III et al.,1991a and 1991b, supra); however, other data suggest the existence ofother protective factors derived from the pituitary.

Very recently, murine macrophage migration inhibitory factor (MIF) wasidentified as an LPS-induced pituitary protein (Bernhagen et al., 1993,J. Cell. Biochem. Supplement 17B, Abstract E306). While it may behypothesized that MIF is one of such protective factors capable ofcounteracting the adverse effects of cytokines in endotoxaemias, itsrole in septic shock had not been investigated prior to the presentinvention.

2.3. MIF: Macrophage Migration Inhibitory Factor

Although MIF was first described over 25 years ago as a T cell productthat inhibits the random migration of guinea pig macrophages (Bloom &Bennett, 1966, Science 158: 80-82; David, 1966, Proc. Natl. Acad. Sci.USA 65: 72-77), the precise role of MIF in either local or systemicinflammatory responses has remained largely undefined. MIF has beenreported to be associated with delayed-type hypersensitivity reactions(Bloom & Bennett, 1966, supra; David, 1966, supra), to be produced bylectin-activated T-cells (Weiser et al., 1981, J. Immunol. 126:1958-1962), and to enhance macrophage adherence, phagocytosis andtumoricidal activity (Nathan et al., 1973, J. Exp. Med. 137: 275-288;Nathan et al., 1971, J. Exp. Med. 133: 1356-1376; Churchill et al.,1975, J. Immunol, 115: 781-785). Unfortunately, many of these studiesused mixed culture supernatants that were shown later to contain othercytokines such as IFN-γ and IL-4 that also have migration inhibitoryactivity (McInnes & Rennick, 1988, J. Exp. Med. 167: 598-611; Thurman etal., 1985, J. Immunol. 134: 305-309).

Recombinant human MIF was originally cloned from human T cells (Weiseret al., 1989, Proc. Natl. Acad. Sci. USA 86: 7522-7526), and has beenshown to activate blood-derived macrophages to kill intracellularparasites and tumor cells in vitro, to stimulate IL-Iβ and TNFαexpression, and to induce nitric oxide synthesis (Weiser et al., 1991,J. Immunol. 147: 2006-2011; Pozzi et al., 1992, Cellular Immunol. 145:372-379; Weiser et al., 1992, Proc. Natl. Acad. Sci. USA 89:8049-8052;Cunha et al., 1993, J. Immunol. 150:1908-1912). Until very recently,however, the lack of a reliable source of purified MIF has continued tohamper investigation of the precise biological profile of this molecule.

3. SUMMARY OF THE INVENTION

The present invention relates to compositions and methods which inhibitthe release and/or biological activity of migration inhibitory factor(MIF). The invention further relates to the uses of such compositionsand methods for the treatment of conditions involving cytokine-mediatedtoxicity, which include, but are not limited to shock, inflammation,graft versus host disease, and/or autoimmune diseases.

The inhibition of MIF activity in accordance with the invention may beaccomplished in a number of ways which include, but are not limited to,the use of MIF binding partners, i.e., factors that bind to MIF andneutralize its biological activity, such as neutralizing anti-MIFantibodies, soluble MIF receptors, MIF receptor fragments, and MIFreceptor analogs; the use of MIF-receptor antagonists, such asanti-MIF-receptor antibodies, inactive MIF analogs that bind but do notactivate the MIF-receptor, small molecules that inhibit MIF release, oralter the normal configuration of MIF, or inhibit productive,MIF/MIF-receptor binding; or the use of nucleotide sequences derivedfrom MIF gene and/or MIF receptor gene, including coding, non-coding,and/or regulatory sequences to prevent or reduce MIF expression by, forexample, antisense, ribozye, and/or triple helix approaches. Any of theforegoing methods may be utilized individually or in combination toinhibit MIF release and/or activity in the treatment of the relevantconditions. Further, such treatment(s) may be combined with othertherapies that (a) inhibit or antagonize initiators of cytokine-mediatedtoxicity (e.g. anti-LPS antibody); (b) inhibit or antagonize toxicparticipants in the endogenous cytokine responses (e.g. anti-TNFα,anti-IL-1, anti-IFN-γ, or IL-1 RA); or (c) themselves inhibit orantagonize cytokine-mediated toxicity (e.g. steroids, glucocorticoids orIL-10).

The present invention is based, in part, on the surprising discoveriesthat, first, administration of MIF in vivo increases mortality ofanimals after challenge with endotoxin and, second, that inhibition ofMIF activity in vivo results in enhanced or prolonged survival aftereither challenge with endotoxin or with TNFα, which challenge wouldotherwise result in death due to shock. Prior to the present invention,no role for MIF in the inflammation/shock response was appreciated.Moreover, the existing evidence and observations indicated, at best,that MIF might play a beneficial role in the treatment ofendotoxin-induced shock. First, MIF was shown to enhance macrophagekilling of intracellular parasites and tumor cells. Second, MIF is anendotoxin-induced protein expressed by the pituitary, an organ which hadpreviously been suggested as a source of protective rather thanexacerbative factors involved in the shock syndrome. In contrast to suchexpectations, the Applicants have discovered that MIF activity actuallyexacerbates endotoxin-induced shock, and that inhibition of MIF activitycan be used to successfully treat otherwise lethal effects ofcytokine-mediated toxicity.

The invention is also based, in part, on the Applicants' discovery ofthe role of MIF in humoral immune responses, which is demonstrated inanimal models by way of a working example. In animals immunized with atest antigen in conjunction with the administration of an anti-MIFantibody, an inhibition of the development of a primary immune responseto the test antigen was observed. These results indicate a means bywhich MIF activity modulates the primary immune response and thereforeanti-MIF treatment could potentially be useful in substantially reducingan undesired immune response, such as autoimmunity and allergy. Incontrast, this observation also indicates the involvement of MIF in aprimary immune response. Thus, MIF may be administered as an adjuvantfor an antigen during immunization of a naive individual to induce anenhanced immune response to the antigen.

The invention is illustrated by working examples which demonstrate thatMIF exacerbates endotoxin-induced shock, and that anti-MIF preventsendotoxin-induced lethality in animal models. In addition, the workingexamples also describe the organ distribution of MIF and its receptor,the identification of a murine MIF receptor, the production of anti-MIFmonoclonal antibodies, and the inhibition of immune responses byanti-MIF antibodies.

4. BRIEF DESCRIPTION OF THE FIGURES

FIG. 1: Graph depicting the schematic profile of TNFα and MIF responsesinvolved in LPS-induced shock.

FIG. 2: Potential ribozyme cleavage sites of MIF. Murine (mmif) [SEQ IDNO:1] and human (hmif) [SEQ ID NO:2] nucleotide sequences are compared.Underlined sequences indicate the potential ribozyme cleavage sites.

FIG. 3: Nucleotide sequence of cDNA encoding murine MIF [SEQ ID NO:3].

FIG. 4: Predicted amino acid sequence homology between murine pituitaryMIF [SEQ ID NO:4] and human Jurkat T-cell MIF [SEQ ID NO:5]. PotentialN-linked glycosylation sites are underlined. mu: murine MIF; hu: humanMIF.

FIG. 5: Inhibition of monocyte migration by recombinant MIF. The effectof various concentrations (0.001-10 μg/ml) of rmuMIF on peripheral humanblood monocyte migration was quantitated in a modified Boyden chamber.The percent migration inhibition relative to buffer controls is plottedversus the logarithm of MIF concentration. Each point depicts the meanof independently performed duplicate determinations.

FIG. 6A: MIF potentiates LPS-induced lethality in BALB/c mice. Resultsrepresent the pooled data from two experiments.

FIG. 6B: Anti-MIF antiserum protects against LPS challenge. Resultsrepresent the pooled data from two experiments.

FIG. 7: Anti-MIF antiserum protects against Gram-positive staphylococcalexotoxin challenge.

FIG. 8: Western blot analysis of MIF released by pituitary cells.

FIGS. 9A and 9B: Immunocytochemistry of MIF release by pituitary cells.FIG. 9.A. Cells cultured in the absence of LPS; FIG. 9.B. Cells culturedin the presence of 25 μg/ml LPS.

FIG. 10 LPS-induced expression of pituitary MIF mRNA in BALB/c mice.

FIG. 11: Competitive PCR quantitation of pituitary MIF mRNA fromLPS-stimulated BALB/c mice.

FIG. 12: Presence of MIF protein in mouse pituitaries.

FIG. 13: Temporal analysis of MIF levels, in sera of mice treated withLPS.

FIG. 14: Western blotting and densitometric analysis of serum MIF inBALB/c, BALB/c^(nu/nu), and hypophysectomized BALB/c mice. Mice wereinjected i.p. with LPS and blood sampled at intervals for Westernblotting analysis. The content of MIF in serum aliquots (5 μl) wasquantified by laser densitometry with reference to electrophoresed rMIFstandards. The inset shows the MIF blot for the BALB/c mice,demonstrating the time-dependent increase in serum MIF after LPSadministration. Each plotted point is the mean±SEM of individual serafrom 2 to 5 animals.

FIG. 15: Western blotting analysis of the MIF content of non-stimulatedmonocytes/macrophages, T-lymphocytes, and PMNs. Ten μl of cell lysate(equivalent to 5×10³ cells) were electrophoresed through 18% gels,transferred to nitrocellulose membrane, and analyzed with anti-MIFpolyclonal antibody. 30 ng of rMIF was electrophoresed and transferredas a standard.

FIG. 16A, 16B, 16C: Western blotting analysis of MIF secretion by RAW264.7 cells (FIG. 16A) and by thioglycollate-elicited peritonealmacrophages stimulated with LPS (FIG. 16B) or with IFNγ plus LPS (FIG.16C). Macrophages (4×10⁶ RAW 264.7 cells or 1×10⁷ peritonealmacrophages) were incubated for 12 h with LPS or IFNγ plus LPS at theindicated concentrations. The content MIF secreted into the medium wasanalyzed by Western blotting. rMIF (30 ng in panel A and 20 ng in panelsB and C) was electrophoresed and transferred as a standard.

FIG. 17: RT-PCT analysis of MIF, TNFα, and β-actin expression byLPS-stimulated RAW 264.7 macrophages. Cells (4×10⁶) were incubated for12 h in medium containing RPMI/1% FBS and LPS at the indicatedconcentrations. Total cellular RNA was extracted, CDNA prepared, andgene-specific PCR products analyzed by agarose gel electrophoresis asdescribed in Materials and Methods.

FIG. 18: Competitive PCR analysis of macrophage MIF mRNA after LPSstimulation. RAW 264.7 cells (4×10⁶) were incubated for 12 h in RPMI/1%FBS with or without LPS (100 pg/ml). RNA was extracted and MIF mRNAlevels analyzed as described in Materials and Methods. The amount of MIFcompetitor template DNA ranged from 0.25 to 15 pg per reaction as shown.

FIG. 19: Western blotting analysis of cytokine-induced MIF secretion byRAW 264.7 macrophages. Cells (4×10⁶) were incubated for 12 h with TNFα,IL-1P, IL-6 (10 or 1 ng/ml), or IFNγ (1000 or 100 IU/ml). 20 ng of rMIFwas electrophoresed and transferred as a standard.

FIG. 20: Concentration of TNFα in cell-culture supernatants of RAW 264.7macrophages stimulated with rMIF. Cells (4×10⁶) were incubated for 12 hwith the indicated concentrations of rMIF or with medium alone(control). Culture medium then was removed and the TNFα contentquantified by L929 cell cytotoxicity. TNFα concentration is expressed asthe difference between the level produced by rMIF-stimulated cells andby control (non-stimulated) cells. Data are expressed as the mean±SD ofthree separate experiments.

FIG. 21: Western blotting analysis of the inhibition by20α-dihydrocortisol of the MIF response of RAW macrophages todexamethasone.

FIG. 22: Western blotting analysis of the organ distribution of murineMIF protein. Organs were obtained from an untreated BALB/c mouse.Aliquots (60 μg of protein) of liver, spleen, kidney, adrenal, brain andlung lysates were electrophoresed through 18% gels, transferred tonitrocellulose membrane, and analyzed with anti-MIF polyclonal antibody.20 ng of rMIF was electrophoresed and transferred as a standard.Although not visible, a MIF band of much lower intensity also wasdetected in the adrenals and lungs.

5. DETAILED DESCRIPTION OF THE INVENTION

The present invention involves compositions and methods that inhibit MIFrelease and/or activity in vivo, for the treatment of any conditionsinvolving cytokine-mediated toxicity, which include but are not limitedto shock, including endotoxin-induced septic shock and exotoxin-inducedtoxic shock, inflammation, graft versus host disease, autoimmunediseases, acute respiratory distress syndrome, granulomatous diseases,chronic infections, transplant rejection, cachexia, viral infections,parasitic infections, including for instance malaria, and/or bacterialinfections.

The inhibition of MIF activity in accordance with the invention may beaccomplished in a number of ways, which may include, but are not limitedto, the use of factors which bind to MIF and neutralize its biologicalactivity; the use of MIF-receptor antagonists; the use of compounds thatinhibit the release of MIF from cellular sources in the body; and theuse of nucleotide sequences derived from MIF coding, non-coding, and/orregulatory sequences to prevent or reduce MIF expression. Any of theforegoing may be utilized individually or in combination to inhibit MIFactivity in the treatment of the relevant conditions, and further, maybe combined with any other anti-cytokine therapy (including steroidtherapy), anti-initiator therapy, inhibitory cytokines or anycombination thereof.

5.1, MIF is a Critical Mediator in the Shock Syndrome and IntegratesCentral and Peripheral Inflammatory Responses

MIF is identified herein as both a neuroendocrine mediator and amacrophage cytokine which plays an important role in host inflammatoryresponses to infection and tissue invasion. Upon release after a proinflammatory stimulus, macrophage-derived MIF may act together with TNFαand other cytokines to coordinate local cellular responses againstinfection or tissue invasion. Pituitary-derived MIF, however, may serveto prime systemic immune responses once a legalized inflammatory siteails to contain an invasive agent, or else act as a CNS-derived stresssignal to activate the immune system against invasion. Thus, MIF may actin concert with adrenocorticotrophic hormone (ACTH) and theadrenocortical axis to modulate systemic inflammatory responses.

The working examples described infra, demonstrate that MIF, historicallyconsidered to be a product of activated T-lymphocytes, is also producedby the pituitary and macrophages (see Section 11 and 12; infra); thatMIF plays a critical role in endotoxin-induced septic shock (caused bygram negative organisms; see Section 7, infra), exotoxin-induced toxicshock syndrome (caused by gram positive organisms; see Section 8,infra), parasite-induced disease responses (see Section 9, infra), andnon-infectious inflammatory diseases, e.g., involving the cell-mediatedimmune response (see Section 10, infra); and that MIF acts in concertwith glucocorticoids to regulate inflammation and immunity (see Section13, infra); and that MIF plays a role in the development of a primaryimmune response (see Section 16, infra). In particular, the results showthat MIF potentiates lethality of endotoxemia, whereas the inhibition ofMIF confers protection against lethal endotoxemia. Inhibition of MIFsimilarly confers protection against toxic shock syndrome. Surprisingly,MIF is induced by many of the glucocorticoids that are considered to beanti-inflammatory agents.

The experiments in animals described herein indicate that a dominantrole of MIF is to serve as a pro inflammatory mediator. A proinflammatory spectrum of action for MIF has been verified by in vitrostudies described herein. As shown in Section 6, infra, recombinant(rMIF) was found to induce TNFα secretion by macrophages, indicatingthat MIF and TNFα may act locally in a reciprocal, proinflammatory loop,i.e., MIF and TNFα stimulate the production of each other by cells ofthe immune system. Recombinant MIF, in combination with otherproinflammatory stimuli, e.g., IFN-γ, also promotes murine macrophagenitric oxide (NO) synthesis (see Section 6, infra).

The Applicants have found that the resting pituitary contains largestores of preformed MIF which are released into the circulation afteractivation of the hypothalamic-pituitary axis by endotoxin, but not frompituicytes by several other cytokines which participate in theproinflammatory response, such as TNFα, IL-β, IL-6 or IFN-γ (see Section11, infra). MIF is also released by macrophages in response to low dosesof endotoxin, and in response to TNFα and IFN-γ (see Section 12, infra),in response to parasitic infection (see Section 9 infra), and inresponse to steroids (see Section 13, infra). Thus, the macrophage isnot only a target for MIF, but is also an important source of MIF invivo.

FIG. 1 depicts the profile of endotoxin-induced MIF and TNFα serumconcentrations detected during the shock response. Detectable serumconcentrations of TNFα quickly peak and clear within 1-2 hours ofexposure to endotoxin. In contrast, detectable serum concentrations ofMIF gradually increase during the acute phase (1-8 hours), peak at 8hours and persist during the post-acute phase (>8 hours) for up to 20hours. While not limited to any theory of operation, the followingcellular sources are proposed for the serum concentrations of MIFobserved, based on the data presented in Sections 11 and 12 infra, andFIGS. 13 and 14: MIF is probably produced by activated T-cells andmacrophages during the proinflammatory stage of endotoxin-induced shock,e.g., as part of the localized response to infection. Once released by apro-inflammatory stimulus, e.g., low concentrations of LPS, or by TNFαand IFN-γ (but not IL-β or IFN-γ), macrophage-derived MIF is theprobable source of MIF produced during the acute phase of endotoxicshock. Both the pituitary, which releases MIF in response to LPS (butnot INF-α, IL-β, IL-6 or IFN-γ), and macrophages are the probable sourceof MIF in the post-acute phase of endotoxic shock, when the infection isno longer confined to a localized site.

In view of Applicants' discovery that the pituitary is a source of MIF,the notion that hypophysectomy sensitizes experimental animals to thelethal effects of LPS initially suggested that MIF may act todown-regulate host inflammatory responses. Therefore, it was surprisingto find that purified, recombinantly prepared MIF (rMIF), when injectedin mice produced visible symptoms of endotoxemia, and when co-injectedwith LPS, potentiated lethality in mice. E.g., see the results presentedin the working example in Section 7, infra, which demonstrate that MIFsubstantially amplifies endotoxin-induced mortality of animals. Thus,contrary to what was expected, it is shown herein that an increase inMIF is not beneficial to the host system, and, in fact, appears toexacerbate cytokine-mediated conditions such as septic shock. Theresults also show that MIF is induced by toxic shock syndrome toxin-1(TSST-1), a staphylococcal exotoxin (Section 8, infra), and by hemozoin,a specific malarial product (Section 9, infra).

It was equally surprising to discover that inhibition of MIF conferredprotection against lethal toxemia. As demonstrated in the workingexample presented in Section 7, infra, inhibition of MIF activity leadsto a dramatic increase in the cumulative survival of animals challengedwith high-dose endotoxin or TNFα. Anti-MIF antiserum also markedlyreduced mortality in test animals challenged with lethal doses of TSST-1(see Section 8, infra). These data establish that MIF contributessignificantly to lethality and may in this sense be considered anecessary or essential component of the shock syndrome.

In accordance with the invention, the inhibition of MIF activity and/orrelease may be used to treat inflammatory response and shock. Beneficialeffects may be achieved by intervention at both early and late stages ofthe shock response. In this respect, the working examples also describethe production of monoclonal antibodies directed against both human andmurine MIF, which may be used to neutralize MIF activity (see Section17, infra). While not limited to any theory or mechanism responsible forthe protective effect of MIF inhibition, preliminary studies show thatanti-MIF therapy is associated with an appreciable (up to 35-40%)reduction in circulating serum TNFα levels. This reduction is consistentwith the TNFα-inducing activity of MIF on macrophages in vitro, andsuggests that MIF is responsible, in part, for the extremely high peakin serum TNFα level that occurs 1-2 hours after endotoxin administrationdespite the fact that MIF cannot be detected in the circulation at thistime. Thus, MIF inhibition therapy may be beneficial at the early stagesof the inflammatory response.

MIF also plays an important role during the post-acute stage of theshock response, and therefore, offers an opportunity to intervene atlate stages where other treatments, such as anti-TNFα therapy, areineffective. The working examples described herein show that MIF isreleased at the post-acute stage of shock, when it is detectable in thecirculation. In the experimental system used, anti-MIF therapy protectedagainst lethal shock in animals challenged with high does of endotoxin(i.e., doses which induce release of pituitary MIF into thecirculation), and in animals challenged with TNFα (i.e., where theexogenous TNFα overrides the beneficial reduction in circulatingendogenous TNFα concentrations achieved using anti-MIF). The ability ofanti-MIF therapy to protect animals challenged with TNFα indicates thatneutralization of MIF during the later, post-acute phase of septic shockis efficacious. The protective effect of the antibody may be attributed,in part, to neutralization of pituitary and macrophage MIF released inthe post-acute phase of septic shock when circulating MIF is readilydetected. Because MIF is a necessary component of the shock syndrome,and the peak of serum MIF expression occurs after that of TNFα, MIFinhibitors may be used to successfully treat cytokine-mediatedconditions even later than the point at which administration of TNFαinhibitors is no longer effective.

Since steroids are potent inhibitors of cytokine production, theireffects were examined on MIF secretion by macrophages and pituitarycells (see Section 13, infra). Surprisingly, steroids were found toinduce rather than inhibit MIF secretion by these cells. The secretionof MIF in response to steroids may reduce the benefit of steroid therapycurrently used to treat inflammation. Therefore, MIF inhibition therapymay be used in conjunction with steroids to treat shock and othercytokine-mediated pathological states, particularly in chronicinflammatory states such as rheumatoid arthritis. Such combinationtherapy may be beneficial even subsequent to the onset of pathogenic orother inflammatory responses. For example, administration of steroidsalone inhibits the TNFα response only if given simultaneously with orbefore LPS challenge. In the clinical setting, the administration ofsteroids subsequent to the onset of septic shock symptoms has proven oflittle benefit (Bone et al., 1987, N. Engl. J. Med. 317: 653-658; Springet al., 1984, N. Engl. J. Med. 311: 1137-1141). Combination steroids/MIFinhibition therapy may be used to overcome this obstacle. In anotherexample, when treating conditions such as rheumatoid arthritis, theadministration of steroids alone can result in the induction of MIF,which may override the inhibitory effects of the administered steroid onthe inflammatory response. In such cases, MIF inhibition therapy can beused in conjunction with steroid treatment. Therapy can be designed toinhibit MIF release and/or activity locally and/or systemically.

Finally, in yet another aspect of the invention, newly identifiedMIF-inhibitors are described. For example, the Applicant's havediscovered that certain steroid derivatives commonly thought to beinactive or thought to block the anti-inflammatory effects of steroids(“anti-steroids”) actually inhibit MIF release in response to subsequentsteroid challenge. Such “inert” or “anti-steroid” compounds may be usedin conjunction with anti-inflammatory steroids to treat inflammatorydiseases, especially non-infectious inflammatory diseases such asautoimmunity, rheumatoid arthritis, graft versus host disease etc. Inparticular, the data described herein (see Section 13, infra) show thatsteroids, such as dexamethasone, which are commonly used to treatinflammation actually induce MIF release. The steroid-induced release ofoccurs at dose ranges that are used in vivo to avoid side effects, e.g.,severe water retention, etc. (i.e., higher doses of steroids whichresult in serious side effects, and lower doses of steroids which areineffective to treat inflammation do not appear to induce MIF release).The release of MIF in response to steroid challenge can be inhibited bypretreating with a MIF-inhibiting dose of the so-called “inactive” or“anti-steroid” derivatives, such as 20α-dihydrocortisol. Because thesederivatives are “inert” high doses which inhibit-MIF release should bewell tolerated. The prior administration of the “inactive” compoundshould not induce side effects, should inhibit MIF release, and thus,may potentiate the anti-inflammatory action of the “active” steroidsubsequently administered. Assays are described herein to identifyadditional compounds that can be used to inhibit the steroid-inducedrelease of MIF from cellular sources in the body.

The following subsections summarize the data presented in sections 6through 15, infra.

5.1.1. Identification of MIF as a Pituitary Hormone

To investigate the production and release of novel pituitary products,we examined the secretory profile of the murine anterior pituitary cellline, AtT-20. These corticotrophic cells have been used widely to studyACTH release and we thought they were potentially useful cells in whichto begin to investigate humoral interactions between the central nervoussystem (CNS), the hypothalamus and the periphery. As shown in Section 9,infra, we incubated AtT-20 cells with LPS for various time intervals andexamined culture supernatants by SDS-PAGE. These analyses revealed thespecific release by pituitary cells of an apparently novel 12.5 kDaprotein. The 12.5 kDa protein was then isolated and determined byamino-terminal sequencing to be highly similar to an amino acid sequencepredicted from a recently cloned human MIF cDNA (Weiser et al., 1989,supra). We cloned this pituitary protein from the cDNA of LPS-stimulatedAtT-20 cells and showed that it is actually the murine homolog of humanMIF (90% identity over 115 amino acids) (see Section 6, infra). Aslittle as 100 pg/ml of LPS was found to induce pituitary cell secretionof MIF. However, pituitary MIF secretion was not induced by TNF-α, IL-β,IL-6 or IFN-γ (see Section 11, infra).

Immunocytochemical analyses revealed that resting, nonstimulatedpituitary cells contain significant amounts of preformed MIF. Thus, alarge fraction of the MIF that is released by pituitary cells inresponse to LPS arises from stored, intracellular pools. Electronmicroscopic studies of whole mouse pituitaries labeled withimmunogold-conjugated anti-MIF antibody localize MIF to granules presentwithin corticotrophic cells. It appears that in vivo, MIF-containinggranules are released either by the direct action of circulatingendotoxin or by specific hypothalamic releasing factor(s).

In vivo studies in mice showed that pituitary MIF mRNA levels increaseafter LPS challenge and reach a plateau after 16-24 hours. Over the sametime course, the pituitary content of preformed MIF protein decreases toalmost undetectable levels. To determine whether the release ofpituitary MIF is associated with a concomitant rise in serum MIF, weobtained blood samples from LPS-injected mice and analyzed serum for MIFcontent by western blotting. In normal mice, MIF was detected 2 hoursafter administration of LPS and its levels increased in serum overapproximately 20 hours. This time course for the rise in serum MIF isconsistent both with the decrease in pituitary MIF protein and with theincrease in pituitary MIF mRNA that occurred after injection of LPS. Incontrast, MIF in the serum of hypophysectomized mice showed a markedlydifferent pattern of induction. Serum specimens obtained fromLPS-treated, hypophysectomized mice showed no detectable MIF at 20hours, the time at which serum MIF levels were highest in normal mice.These data are consistent with the notion that the pituitary is criticalto the MIF response that appears in serum during endotoxemia, and thatpituicytes contribute to serum levels of MIF. We then calculated theamount of preformed MIF stored in the pituitary and the amount of MIFthat appears in serum after activation by LPS of thehypothalamic-pituitary axis. The quantities of MIF present in thepituitary and in serum were well within the range observed for ACTH andother anterior pituitary hormones (Table I).

TABLE I COMPARISON OF PITUITARY MACROPHAGE MIGRATION INHIBITORY FACTOR(MIF) CONTENT AND SERUM MIF LEVELS WITH LEVELS OF OTHER ANTERIORPITUITARY HORMONES Pituitary content^(a) Serum level^(b) Hormone (% oftotal protein) (ng ml⁻¹⁾ MIF 0.05  80-340 ACTH 0.2  2-500 Prolactin 0.08200-300 ^(a)Pituitary hormone content at resting conditions ^(b)Serumhormone level under conditions of maximal pituitary stimulation.

5.1.2. Macrophage MIP Production

As shown in Section 12, infra, hypophysectomized mice have a markedlyabnormal serum MIP response compared with control mice. The gradualincrease in serum MIF over 20 hours is not observed. Instead, on closerexamination, a peak of serum MIF is observed during the early, acutephase of endotoxaemia (≦3 h after LPS injection). Given the lack of apituitary in hypophysectomized mice, and the well-acceptedunresponsiveness of T cells to LPS, we considered that the MIF presentin the circulation at 2 hours must reflect the release of this cytokineby an additional LPS-sensitive cell population. Based on the observationthat such an early peak in the serum MIF response is reminiscent of amacrophage TNFα response, we examined the possibility that MIF is alsoproduced by cells of the monocyte-macrophage lineage.

Significant amounts of preformed MIF protein were found in the resting,nonstimulated murine monocyte cell line RAW 264.7, murine peritonealmacrophages and the human monocyte cell line THP-1. Western blottingrevealed that the MIP content of murine or human monocytes-macrophageswas similar to that of the murine ASL-1 and the human Jurkat T celllines. In contrast, cell lysates obtained from purifiedpolymorphonuclear leukocytes did not contain detectable MIF protein.Whole tissue analyses demonstrated significant amounts of both MIFprotein and mRNA in organs that have a high content of macrophages (seeSection 14, infra), complementing recent observations that MIF mRNA ispresent constitutively in tissues such as the spleen, the liver, and thekidney. (Lanahan et al., 1992, Mol. Cell. Biol. 12: 3919-3929).

Various proinflammatory stimuli, such as LPS, TNFα and IFN-γ wereobserved to be potent inducers of macrophage MIF release (see Section12, infra). Secretion of significant amounts of MIF occurred at LPSconcentrations (10-100 pg ml⁻¹) that are lower in general than has beenobserved to be optimal for the induction of TNFα release. Significantamounts of preformed MIF mRNA were also present in resting macrophages,and stimulation by LPS increased these mRNA levels approximatelytwo-fold. Peak mRNA stimulation also occurred at very low concentrationsof LPS (1 pg ml⁻¹).

5.1.3. Cloning, Expression and Biochemical Characterization of MIF

The cloning of murine MIF (muMIF) from an anterior pituitary cell lineand the purification of native MIF from mouse liver are described insection 6, infra. For comparison purposes, human MIF (huMIF) was clonedfrom the Jurkat T-cell line. Sequence analysis of murine pituitary MIFand human T-cell MIF cDNA demonstrated a 90% homology between the murineand human proteins. Cloned Jurkat T-cell MIF cDNA differs by onenucleotide from the first published human T-cell MIF cDNA (Weiser etal., 1989), but is in agreement with human MIF cDNA derived from humanlens tissue (Wistow et al., 1993, Proc. Natl. Acad. Sci. USA 90:1272-1275), and with a T-cell glycosylation inhibition factor (GIF) cDNA(Mikayama et al., 1993, Proc. Natl. Acad. Sci. USA 90: 10056-10060). Thesingle nucleotide difference results in a change in ˜he deduced aminoaCld sequence (Ser 106→Asn 106), thus increasing homology of the humanand murine proteins.

DNA homology analysis showed that both human and murine MIF lack aconventional N-terminal leader sequence. Thus, MIF joins a growing listof cytokines, such as IL-1 (Rubartelli et al., 1990, EMBO J. 9:1503-1510), basic fibroblast growth factor (bFGF; Jackson et al., 1992,Proc. Natl. Acad. Sci. USA 89: 10691-10695), and a secreted form ofcyclophilin (Sherry et al., 1992, Proc. Natl. Acad. Sci. USA 89:3511-3515) which are released from cells by non-classical proteinsecretion pathways.

Recombinant murine and human MIF were expressed in E. coli, purified tohomogeneity by a simple two-step procedure, and the proteinscharacterized by a variety of biochemical and biological criteria.Native MIF obtained from mouse liver was found to be monomeric and to befree of significant post-translational modifications as assessed both byendoglycosidase F treatment followed by SDS-PAGE/Western blotting and bymass spectroscopic analysis. Recombinant and native MIF exhibited anumber of comparable effects on monocytes/macrophages. Recombinant MIFwas found to inhibit monocyte migration when assayed in modified Boydenchambers. MIF also induced TNFα secretion, and promoted the release ofnitric oxide (NO) from macrophages primed with IFN-γ. Circular dichroism(CD) spectroscopy revealed that bioactive, recombinant MIF exhibits ahighly ordered three-dimensional structure, with a >55% content ofβ-pleated sheet and α-helix. Thermodynamic stability studies also showedthat despite a high content of ordered secondary structure elements, theconformation of MIF was readily perturbed by denaturing solventconditions. These studies define a number of the biochemical andbiological properties of recombinant and purified, native MIF andprovide, for the first time, information concerning thethree-dimensional structure of this protein.

5.1.4. Biodistribution of MIF and MIF Receptors

The working example in Section 14, infra, presents a study thatillustrates, the distribution of MIF and MIF-binding sites, i.e.MIF-receptors within the body. The study indicates that MIF receptorsare present in kidney cells, for example. Further, two such MIFreceptors are identified, for the first time, in the working examplepresented, infra, in Section 13. Such receptors may be geneticallyengineered for use as MIF inhibitors.

5.2. Inhibitors of MIF Activity

Described below are factors which may be used as MIF antagonists, i.e.,factors which inhibit the biologic activity of MIF.

5.2.1. MIF-Binding Partners

Factors that bind MIF and neutralize its biological activity,hereinafter referred to as MIF binding partners, may be used inaccordance with the invention as treatments of conditions involvingcytokine-mediated toxicity. While levels of MIF protein may increase dueto endotoxin challenge, the interaction of inhibitory MIF-bindingpartners with MIF protein prohibits a concomitant increase in MIFactivity. Such factors may include, but are not limited to anti-MIFantibodies, antibody fragments, MIF receptors, and MIF receptorfragments.

Various procedures known in the art may be used for the production ofantibodies to epitopes of recombinantly produced (e.g., usingrecombinant DNA techniques described infra), or naturally purified MIF.Neutralizing antibodies, i.e. those which compete for or stericallyobstruct the binding sites of the MIF receptor are especially preferredfor diagnostics and therapeutics. Such antibodies include but are notlimited to polyclonal, monoclonal, chimeric, single chain, Fab fragmentsand fragments produced by an Fab expression library.

For the production of antibodies, various host animals may be immunizedby injection with MIF and/or a portion of MIF. Such host animals mayinclude but are not limited to rabbits, mice, and rats, to name but afew. Various adjuvants may be used to increase the immunologicalresponse, depending on the host species, including but not limited toFreund's (complete and incomplete), mineral gels such as aluminumhydroxide, surface active substances such as lysolecithin, pluronicpolyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanin,dinitrophenol, and potentially useful human adjuvants such as BCG(bacille Calmette-Guerin) and Corynebacterium parvum.

Monoclonal antibodies to MIF may be prepared by using any techniquewhich provides for the production of antibody molecules by continuouscell lines in culture. These include but are not limited to thehybridoma technique originally described by Kohler and Milstein,(Nature, 1975, 256:495-497), the human B-cell hybridoma technique(Kosbor et al., 1983, Immunology Today, 4:72; Cote et al., 1983, Proc.Natl. Acad. Sci., 80:2026-2030) and the EBV-hybridoma technique (Cole etal., 1985, Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc.,pp. 77-96). In addition, techniques developed for the production of“chimeric antibodies” (Morrison et al., 1984, Proc. Natl. Acad. sci.,81:6851-6855; Neuberger et al., 1984, Nature, 312:604-608; Takeda etal., 1985, Nature, 314:452-454) by splicing the genes from a mouseantibody molecule of appropriate antigen specificity together with genesfrom a human antibody molecule of appropriate biological activity can beused. Alternatively, techniques described for the production of singlechain antibodies (U.S. Pat. No. 4,946,778) can be adapted to produceMIF-specific single chain antibodies. The hybridoma technique has beenutilized to generate anti-MIF monoclonal antibodies. Hybridomassecreting IgG monoclonal antibodies directed against both human andmurine forms of MIF have been isolated and characterized for theirability to neutralize MIF biological activity. Anti-MIF monoclonalantibodies were shown to inhibit the stimulation of macrophagekilling ofintracellular parasites. The anti-MIF monoclonal antibodies have alsobeen utilized to develop a specific and sensitive ELISA screening assayfor MIF. Both the anti-MIF monoclonal antibodies and the ELISA assay maybe used in the diagnosis and/or treatment of inflammatory responses andshock.

Antibody fragments which recognize specific MIF epitopes may begenerated by known techniques. For example, such fragments include butare not limited to: the F(ab′)₂ fragments which can be produced bypepsin digestion of the antibody molecule and the Fab fragments whichcan be generated by reducing the disulfide bridges of the F(ab′)₂fragments. Alternatively, Fab expression libraries may be constructed(Huse et al., 1989, Science, 246:1275-1281) to allow rapid and easyidentification of monoclonal Fab fragments with the desired specificityto MIF.

MIF receptors, MIF receptor fragments, and/or MIF receptor analogs may,in accordance with the invention, be used as inhibitors of MIFbiological activity. By binding to MIF protein, these classes ofmolecules may inhibit the binding of MIF to cellular MIF receptors, thusdisrupting the mechanism by which MIF exerts its biological activity.Small organic molecules which mimic the activity of such molecules arealso within the scope of the present invention.

MIF receptors may include any cell surface molecule that binds MIF in anamino acid sequence-specific and/or structurally-specific fashion. SuchMIF receptors include, but are not limited to the 72 kD MIF receptorclass having a partial amino acid sequence of:

AKKGAVGGI [SEQ ID NO: 6]and the 52 kD receptor having a partial amino acid sequence of:

I-X-HNTVATEI(S)(G)YN(N/G)A(M) [SEQ ID NO: 7]both of which are presented in the working example in Section 13, infra.The residues in parenthesis are provisional assignment.

Additional MIF receptors and genes that encode MIF receptors may beidentified, isolated, and cloned using a variety of techniques wellknown to those of ordinary skill in the art. For example, MIF receptormolecules may be identified and isolated using standard affinitychromatography techniques wherein those molecules exhibiting sequence-and/or structural binding specificity to MIF protein are separated fromother non-MIF binding molecules. The MIF binding proteins may beadditionally purified, using standard techniques, at which point theprotein may be tested and utilized for its ability to inhibit MIF.

Alternatively, the amino acid sequence of the purified protein may be atleast partially determined, and then used to design oligonucleotideswith which to screen cDNA and/or genomic libraries in order to clone thegene(s) that encode(s) MIF receptors, techniques of which are well knownto those of skill in the art. Further, new MIF receptor genes may becloned by construction of a cDNA library in a mammalian expressionvector such as pcDNA1, that contains SV40 origin of replicationsequences which permit high copy number expression of plasmids whentransferred into COS cells. The expression of the MIF receptor on thesurface of transfected COS cells may be detected in a number of ways,including the use of radioactive, fluorescent, or enzymatically labeledMIF. Cells expressing an MIF receptor may be enriched by subjectingtransfected cells to a FACS (fluorescent activated cell sorter). For areview of cloning strategies which may be used, see e.g., Maniatis,1989, Molecular Cloning, A Laboratory Manual, Cold Springs Harbor Press,N.Y.; and Ausubel et al., 1989, Current Protocols in Molecular Biology,(Green Publishing Associates and Wiley Interscience, N.Y.)

Fragments of any of the MIF receptors described above may also be usedas MIF inhibitory agents, and any MIF receptor fragment possessing anyamino, carboxy, and/or internal deletion that specifically binds MIF soas to inhibit MIF biological activity is intended to be within the scopeof this invention. An amino and/or carboxy deletion refers to a moleculepossessing amino and/or carboxy terminal truncations of at least oneamino acid residue. An internal deletion refers to molecules thatpossess one or more non-terminal deletions of at least one amino acidresidue. Among these MIF receptor fragments are truncated receptors inwhich the cytoplasmic or a portion of the cytoplasmic domain has beendeleted, and fragments in which the cytoplasmic and the transmembranedomain(s) has been deleted to yield a soluble MIF receptor containingall or part of the MIF receptor extracellular domain.

MIF receptor analogs which specifically bind MIF may also be used toinhibit MIF activity. Such MIF receptor analogs may include MIF receptoror receptor fragments further possessing one or more additional aminoacids located at the amino terminus, carboxy terminus, or between anytwo adjacent MIF receptor amino acid residues. The additional aminoacids may be part of a heterologous peptide functionally attached to allor a portion of the MIF receptor protein to form a MIF receptor fusionprotein. For example, and not by way of limitation, the MIF receptor, ora truncated portion thereof, can be engineered as a fusion protein witha desired Fc portion of an immunoglobulin. MIF receptor analogs may alsoinclude MIF receptor or MIF receptor fragments further possessing one ormore amino acid substitutions of a conservative or non-conservativenature. Conservative amino acid substitutions consist of replacing oneor more amino acids with amino acids of similar charge, size, and/orhydrophobicity characteristics, such as, for example, a glutamic acid(E) to aspartic acid (D) amino acid substitution. Non-conservativesubstitutions consist of replacing one or more amino acids with aminoacids possessing dissimilar charge, size, and/or hydrophobicitycharacteristics, such as, for example, a glutamic acid (E) to valine (V)substitution.

The MIF receptors, MIF receptor fragments and/or 35 analogs may be madeusing recombinant DNA techniques. Here, the nucleotide sequencesencoding the peptides of the invention may be synthesized, and/orcloned, and expressed according to techniques well known to those ofordinary skill in the art. See, for example, Ausubel, F. M. et al.,eds., 1989, Current Protocols In Molecular Biology, Vol. 1 and 2, JohnWiley and Sons, New York. Caruthers, et al., 1980, Nuc. Acids Res. Symp.Ser. 7:215-233; Crea and Horn, 1980, Nuc. Acids Res. 9(10):2331;Matteucci and Caruthers, 1980, Tetrahedron Letters 21:719; and Chow andKempe, 1981, Nuc. Acids Res. 9(12):2807-2817.

Alternatively, the protein itself could be produced using chemicalmethods to synthesize the amino acid sequence in whole or in part. Forexample, peptides can be synthesized by solid phase techniques, cleavedfrom the resin, and purified by preparative high performance liquidchromatography. (E.g., see Creighton, 1983, Proteins Structures AndMolecular Principles, W.H. Freeman and Co., N.Y. pp. 50-60.) Thecomposition of the synthetic peptides may be confirmed by amino acidanalysis or sequencing (e.g., the Edman degradation procedure; seeCreighton, 1983, Proteins, Structures and Molecular Principles, W.H.Freeman and Co., N.Y., pp. 34-49).

These molecules may also be synthesized utilizing alternative procedureswhich may advantageously affect certain of the molecules' properties,such as stability, bioavailability, and MIF inhibitory activity. Forexample, MIF receptors, MIF receptor fragments, and MIF receptor analogsmay be synthesized such that one or more of the bonds which link theamino acid residues of the peptides are non-peptide bonds. Thesealternative non-peptide bonds may be formed by utilizing reactions wellknown to those in the art, and may include, but are not limited toimino, ester, hydrazide, semicarbazide, and azo bonds, to name but afew. In another embodiment, the proteins may be synthesized withadditional chemical groups present at their amino and/or carboxytermini. For example, hydrophobic groups such as carbobenzoxyl, dansyl,or t-butyloxycarbonyl groups, may be added to the peptides' aminoterminus. Further, the peptides may be synthesized such that theirsteric configuration is altered. For example, the D-isomer of one ormore of the amino acid residues of the peptide may be used, rather thanthe usual L-isomer.

5.2.2. MIF-Receptor Antagonists

Molecules which inhibit MIF biological activity by binding to MIFreceptors may also be utilized for the treatment of conditions involvingcytokinemeditated toxicity. Such molecules may include, but are notlimited to anti-MIF receptor antibodies and MIF analogs.

Anti-MIF receptor antibodies may be raised and used to neutralize MIFreceptor function. Antibodies against all or any portion of a MIFreceptor protein may be produced, for example, according to thetechniques described in Section 5.2.1., supra.

MIF analogs may include molecules that bind the MIF receptor but do notexhibit biological activity. Such analogs compete with MIF for bindingto the MIF receptor, and, therefore, when used in vivo, may act to blockthe effects of MIF in the progress of cytokine-mediated toxicity. Avariety of techniques well known to those of skill in the art may beused to design MIF analogs. The coding sequence for human MIF describedherein, differs from the published sequence, resulting in a serine toasparagine change in the gene product at amino acid residue number 106.Both the corrected human sequence and that of the murine protein isdescribed in Section 6, infra, and shown in FIG. 2 [SEQ ID Nos: 1 and2]. Recombinant DNA techniques may be used to produce modified MIFproteins containing, for example, amino acid insertions, deletionsand/or substitutions which yield MIF analogs with receptor bindingcapabilities, but no biological activity. Alternatively, MIF analogs maybe synthesized using chemical methods such as those described above inSection 5.2.1. (see, for example, Sambrook et al., 1989, MolecularCloning, A Laboratory Manual, Cold Spring Harbor Press, N.Y.).

MIF receptors and/or cell lines that express MIF receptors may be usedto identify and/or assay potential MIF antagonists. For example, onemethod that may be pursued in the identification of such MIF antagonistmolecules would comprise attaching MIF receptor molecules to a solidmatrix, such as agarose or plastic beads, microtiter wells, or petridishes, using techniques well known to those of skill in the art, andsubsequently incubating the attached MIF receptor molecules in thepresence of a potential MIF analog or analogs. After incubating, unboundcompounds are washed away, and the MIF receptor-bound compounds arerecovered. In this procedure, large numbers of types of molecules may besimultaneously screened for MIF receptor-binding activity. Boundmolecules may be eluted from the MIF receptor molecules by, for example,competing them away from the MIF receptor molecules with the addition ofexcess MIF, changing the pH or osmotic strength of the buffer or addingchaotropic agents. The binding/elution steps bring about a partialpurification of the molecules of interest.

In order to continue the purification process, the eluted molecules maybe further fractionated by one or more chromatographic and/or otherseparation techniques well known in the art until the molecule(s) ofinterest is (are) purified to the extent necessary. Once compoundshaving MIF-receptor binding capabilities are identified, the compoundsmay be assayed for their effects on cytokine-mediated toxicity usingcell lines such as those described in this Section, or by normalexperimental animal models or alternatively, by utilizing transgenicanimal models such as those described in Section 5.4, infra.

Alternatively, screening of peptide libraries with recombinantlyproduced MIF receptors and/or MIF receptor fragments may be used toidentify potential MIF analogs. Once peptides that bind MIF receptor areidentified using this screening technique, their effects oncytokine-mediated toxicity may be assayed using cells lines such asthose described in this Section, or alternatively, may be evaluatedusing normal experimental animal models or transgenic animals such asthose described in Section 5.4., infra. Small organic molecules whichmimic the activity of such peptides are also within the scope of thepresent invention.

Random peptide libraries consist of all possible combinations of aminoacids, and such libraries may be attached to a suitable smallparticulate solid phase support and used to identify peptides that areable to bind to a given receptor (Lam, K. S. et al., 1991, Nature 354:82-84). The screening of peptide libraries may have therapeutic value inthe discovery of pharmaceutical agents that act to inhibit thebiological activity of receptors through their interactions with thegiven receptor.

Identification of molecules that are able to bind to the MIF receptormay be accomplished by screening a peptide library with recombinantsoluble MIF receptor protein. Methods for expression and purification ofmolecules such as MIF receptors are well known to those of skill in theart. For screening, it is preferred to label or “tag” the MIF receptormolecule. The protein may be conjugated to enzymes such as alkalinephosphatase or horseradish peroxidase or to other reagents such asfluorescent labels which may include fluorescein isothyiocynate (FITC),phycoerythrin (PE) or rhodamine. Conjugation of any given label to theMIF receptor may be performed using techniques that are routine in theart. Alternatively, MIF receptor expression vectors may be engineered toexpress a chimeric MIF receptor protein containing an epitope for whicha commercially available antibody exists. The epitope-specific antibodymay be tagged using methods well known in the art including labelingwith enzymes, fluorescent dyes or colored or magnetic beads.

The “tagged” MIF receptor or receptor/conjugate is incubated with therandom peptide library for 30 minutes to one hour at 22° C. to allowcomplex formation between MIF receptor and peptide species within thelibrary. The library is then washed to remove any unbound MIF receptorprotein. If MIF receptor has been conjugated to alkaline phosphatase orhorseradish peroxidase the whole library is poured into a petri dishcontaining a substrates for either alkaline phosphatase or peroxidase,for example, 5-bromo-4-chloro-3-indoyl phosphate (BCIP) or3,3′,4,4″-diaminobenzidine (DAB), respectively. After incubating forseveral minutes, the peptide/solid phase-MIF receptor complex changescolor, and can be easily identified and isolated physically under adissecting microscope with a micromanipulator. If a fluorescent taggedMIF receptor molecule has been used, complexes may be isolated byfluorescence-activated sorting. If a chimeric MIF protein expressing aheterologous epitope has been used, detection of the peptide/MIFreceptor complex may be accomplished by using a labeled epitope-specificantibody. Once isolated, the MIF receptor conjugate may be eluted off,the peptide support washed, and the identity of the peptide attached tothe solid phase support determined by peptide sequencing.

MIF analogs may also be identified using cell lines that express MIFreceptor. Such cell lines may be ones which naturally express thereceptor, such as RAW 264.7 cells, or alternatively, cell lines thathave been engineered using recombinant techniques to express MIFreceptor. These cell lines may also be used to evaluate potential MIFanalogs identified using MIF receptor binding techniques such as thosedescribed above.

With respect to engineered cell lines, a variety of cells may beutilized as host cells for expression of the recombinant MIF receptor,including, but not limited to animal cell systems infected withrecombinant virus expression vectors (e.g., adenovirus, vaccinia virus)including cell lines engineered to contain multiple copies of the MIFreceptor DNA either stably amplified (e.g., CHO/dhfr) or unstablyamplified in double-minute chromosomes (e.g., murine cell lines). Incases where an adenovirus is used as an expression vector, the MIFreceptor coding sequence may be ligated to an adenovirustranscription-translation control complex, e.g., the late promoter andtripartite leader sequence. This chimeric gene may then be inserted inthe adenovirus genome by in vitro or in vivo recombination. Insertion ina non-essential region of the viral genome (e.g., region E1 or E3) willresult in a recombinant virus that is viable and capable of expressingMIF receptor in infected hosts (e.g., See Logan & Shenk, 1984, Proc.Natl. Acad. Sci. (USA) 81:3655-3659). Alternatively, the vaccinia 7.5Kpromoter may be used. (See, e.g., Mackett et al., 1982, Proc. Natl.Acad. Sci. (USA) 79: 7415-7419; Mackett et al., 1984, J. Virol. 49:857-864; Panicali et al., 1982, Proc. Natl. Acad. Sci. 79: 4927-4931).

Specific initiation signals may also be required for efficienttranslation of inserted MIF receptor coding sequences. These signalsinclude the ATG initiation codon and adjacent sequences. In cases wherethe entire MIF receptor gene, including its own initiation codon andadjacent sequences, is inserted into the appropriate expression vector,no additional transcriptional control signals may be needed. However, incases where only a portion of the MIF receptor coding sequence isinserted, exogenous transcriptional control signals, including the ATGinitiation codon, must be provided. Furthermore, the initiation codonmust be in phase with the reading frame of the MIF receptor codingsequence to ensure translation of the entire insert. These exogenoustranslational control signals and initiation codons can be of a varietyof origins, both natural and synthetic. The efficiency of expression maybe enhanced by the inclusion of appropriate transcripticn enhancerelements, transcription terminators, etc. (see Bittner et al., 1987,Methods in Enzymol. 153: 516-544).

In addition, a host cell strain may be chosen which modulates theexpression of the inserted sequences, or modifies and processes the geneproduct in the specific fashion desired. Such modifications (e.g.,glycosylation) and processing (e.g., cleavage) of protein products maybe important for the function of the protein. Different host cells havecharacteristic and specific mechanisms for the posttranslationalprocessing and modification of proteins. Appropriate cells lines or hostsystems can be chosen to ensure the correct modification and processingof the foreign protein expressed. To this end, eukaryotic host cellswhich possess the cellular machinery for proper processing of theprimary transcript, and for any normal glycosylation, and/orphosphorylation of the gene product may be used. Such mammalian hostcells include but are not limited to CHO, VERO, BHK, HeLa, COS, MDCK,293, WI38, etc.

The cell lines may be utilized to screen and identify MIF analogs.Synthetic compounds, natural products, and other sources of potentiallybiologically active materials can be screened in a number of ways by,for example, testing a compound's ability to inhibit binding of MIF to aMIF receptor. Standard receptor binding techniques may be utilized forthis purpose.

The ability of anti-MIF receptor antibodies and potential MIF analogs toreduce or inhibit MIF biological activity may be assayed in vivo byutilizing animals expressing MIF receptor, for instance, normal animals.Such animals may also include transgenic animal models such as thosedescribed below in Section 5.4, infra.

5.2.3. Other Inhibitors of MIF Activity

As explained in Section 5.1, supra, certain steroids, commonly thoughtto be either inactive or “anti-steroidal” actually inhibit the releaseof MIF; 20α dihydrocortisol. These steroids, or any other compound whichinhibits the release of preformed MIF, can be used in combinationtherapy with antiinflammatory steroids.

Compounds which inhibit the release of MIF can be identified in cellbased assays, such as the one described in Section 13, infra. Ingeneral, any pituitary or macrophage cell line that releases MIF inresponse to a challenge dose of steroid can be used. The assay can beconducted by adding the test compound to the cells in culture which arethen challenged with a dose of steroid known to induce MIF release. Testcompounds may be administered simultaneously with, or up to severalhours before or after the challenge dose so as to identify agents thatare useful in inhibiting the MIF response at different stages, i.e.,inhibiting release of pre-formed MIF, versus inhibiting de novosynthesis and release, versus inhibiting both.

The conditioned media is then collected from the cultured cells andassayed for MIF; e.g., by immunoassay, including but not limited to anELISA, Western blot, radioimmunoassay, etc. A reduced amount of MIF inthe conditioned media indicates that the test compound inhibits thesteroid-induced release of MIF. Compounds so identified in this assaymay be used in combination therapy with steroids to treat inflammation.“Biologically inert” or innocuous compounds, such as the inactivesteroids, or steroids which can be used at doses that do not causeundesired side effects, may be preferred for therapeutic use. However,any inhibitory compounds having a good therapeutic index, e.g., lowtoxicity and little or no side effects may be used.

5.2.4. Dose and Treatment Regimens

Inhibitors of MIF biological activity such as anti-MIF antibodies, MIFreceptors, MIF receptor fragments, MIF receptor analogs, anti-MIFreceptor antibodies, MIF analogs and inhibitors of MIF release, may beadministered using techniques well known to those in the art.Preferably, agents are formulated and administered systemically.Techniques for formulation and administration may be found in“Remington's Pharmaceutical Sciences”, 18th ed., 1990, Mack PublishingCo., Easton, Pa. Suitable routes may include oral, rectal, transmucosal,or intestinal administration; parenteral delivery, includingintramuscular, subcutaneous, intramedullary injections, as well asintrathecal, direct intraventricular, intravenous, intraperitoneal,intranasal, or intraocular injections, just to name a few. Mostpreferably, administration is intravenous. For injection, the agents ofthe invention may be formulated in aqueous solutions, preferably inphysiologically compatible buffers such as Hanks' solution, Ringer'ssolution, or physiological saline buffer. For transmucosaladministration, penetrants appropriate to the barrier to be permeatedare used in the formulation. Such penetrants are generally known in theart.

Effective concentrations and frequencies of dosages of the MIFinhibitory compounds invention to be administered may be determinedthrough procedures well known to those in the art, which address suchparameters as biological half-life, bioavailability, and toxicity. Inthe case of anti-MIF antibodies, a preferred dosage concentration mayrange from about 0.1 mg/kg body weight to about 20 mg/kg body weight,with about 10 mg/kg body weight being most preferred. Because antibodiestypically exhibit long half-lives in circulation, a singleadministration of antiserum may be sufficient to maintain the requiredcirculating concentration. In the case of compounds exhibiting shorterhalf-lives, multiple doses may be necessary to establish and maintainthe requisite concentration in circulation.

MIF inhibitors may be administered to patients alone or in combinationwith other therapies. Such therapies include the sequential orconcurrent administration of inhibitors or antagonists of initiators ofcytokine-mediated toxicity (e.g. anti-LPS), inhibitors or antagonists ofparticipants in the endogenous cytokine responses (e.g. anti-TNFα,anti-IL-1, anti-IFN-γ, IL-1 RA); and compounds that inhibit orantagonize cytokine-mediated toxicity directly (e.g. steroids,glucocorticoids or IL-10). MIF inhibitor dosage concentration andfrequency may be altered when used as a part of a combination therapyand, therefore appropriate tests must be performed in order to determinethe best dosage when more than one class of inhibitory compounds is tobe administered.

Because MIF expression reaches a peak level in response to endotoxinchallenge later than TNFα, anti-MIF compounds may be administered afterthe period within which anti-TNFα inhibitors are effective. As shown inthe working example, described in Section 7, infra, anti-MIF antibodyconferred full protection against endotoxemia in animals. Anti-MIF hasalso been shown in Applicants' pilot studies to reduce circulating TNFαlevels, indicating that anti-MIF inhibits the proinflammatory spectrumof activity of MIF, and that anti-MIF inhibits the inflammatory cytokinecascade generally. In in vivo experiments, moreover, anti-MIF was shownto protect against lethal shock induced by administration of exogenousTNFα (see Section 7, infra). Therefore, the beneficial effect of theanti-MIF antibody probably resides in its ability to neutralize thebioactivity of both macrophage MIF released during the acute phase inresponse to a proinflammatory stimulus (e.g., LPS, or TNFα and IFN-γ),and MIF released by both the pituitary and macrophages during thepost-acute phase of the shock response.

The development of anti-MIF monoclonal antibodies provides a specificmeans for disrupting the mechanism by which MIF exerts its biologicalactivity. The anti-MIF monoclonal antibodies may be used as atherapeutic for conditions involving MIF-mediated adverse effectsgenerally, for instance endotoxin lethality and cytokine-mediatedlethality, including TNFα toxicity, such as observed during septicshock. The same antibodies may also be used to protect against the toxiceffects of nitric oxide production by macrophages, which is also inducedby MIF. MIF has been shown to be an important mediator in the immuneresponse to malaria infection, therefore anti-MIF monoclonal antibodiesmay be effective in ameliorating the lethality of parasite-inducedcytokine release. Aside from being an important mediator of theinflammatory immune response, MIF has also been shown to be involved inthe development of a primary immune response. Furthermore, theadministration of anti-MIF monoclonals has been shown to abrogate anantigen-specific immune response, confirming that anti-MIF antibodiesmay be useful therapeutic agents for substantially reducing an undesiredimmune reaction, such as allergy or autoimmunity.

5.3. Inhibitors of MIF and/or MIF Receptor Gene Expression

Nucleotide sequences derived from the coding, non-coding, and/orregulatory sequences of the MIF and/or MIF receptor genes may be used toprevent or reduce the expression of these genes, leading to a reductionor inhibition of MIF activity. The nucleotide sequence encoding thehuman MIF protein has been reported. In addition, as presented in theworking example in Section 6, infra, the nucleotide sequence of humanMIF has been corrected, and a cDNA corresponding to the nucleotidesequence encoding the murine MIF protein has now been identified.Further, the MIF receptor amino acid sequence provided in the workingexample in Section 15, infra, may, for example, be used to designoligonucleotides for the regulation of MIF receptor genes. Among thetechniques by which such regulation of gene expression may beaccomplished are, as described below, antisense, triple helix, andribozyme approaches. Administration of these nucleotide sequences,therefore, may be used to temporarily block expression and/ortranscription of the MIF and/or MIF receptor genes as one method oftreatment for conditions involving cytokine-mediated toxicity.

These approaches which target gene expression may be used alone, incombination with each other, or alternatively, in conjunction with anyof the inhibitory MIF-binding and/or MIF receptor antagonist proceduresdescribed above. Further, these gene regulation approaches may be usedtogether with ant-TNFα, anti-initiators and/or other anti-cytokinetherapies.

5.3.1. Anti-Sense RNA and Ribozymes

Within the scope of the invention are oligoribonucleotide sequences,that include anti-sense RNA and DNA molecules and ribozymes thatfunction to inhibit the translation of MIF and/or MIF receptor mRNA.Anti-sense RNA and DNA molecules act to directly block the translationof mRNA by binding to targeted mRNA and preventing protein translation,either by inhibition of ribosome binding and/or translocation or bybringing about the nuclease degradation of the mRNA molecule itself.

Ribozymes are enzymatic RNA molecules capable of catalyzing the specificcleavage of RNA. The mechanism of ribozyme action involves sequencespecific hybridization of the ribozyme molecule to complementary targetRNA, followed by a endonucleolytic cleavage. Within the scope of theinvention are engineered hammerhead motif ribozyrne molecules thatspecifically and efficiently catalyze endonucleolytic cleavage of MIFand/or MIF receptor mRNA sequences.

Specific ribozyme cleavage sites within any potential RNA target areinitially identified by scanning the target molecule for ribozyrnecleavage sites which include the following sequences GUA, GUU and GUC(See FIG. 2 for an illustration of such potential sites on murine andhuman MIF cDNA). Once identified, short RNA sequences of between 15 and20 ribonucleotides corresponding to the region of the target genecontaining the cleavage site may be evaluated for predicted structuralfeatures such as secondary structure that may render the oligonucleotidesequence unsuitable. The suitability of candidate targets may also beevaluated by testing their accessibility to hybridization withcomplimentary oligonucleotides, using ribonuclease protection assays.

Both anti-sense RNA and DNA molecules and ribozymes of the invention maybe prepared by any method known in the art for the synthesis of nucleicacid molecules. These include techniques for chemically synthesizingoligodeoxyribonucleotides well known in the art such as, for example,solid phase phosphoramidite chemical synthesis. Alternatively, RNAmolecules may be generated by in vitro and in vivo transcription of DNAsequences encoding the antisense RNA molecule. Such DNA sequences may beincorporated into a wide variety of vectors which incorporate suitableRNA polymerase promoters such as the T7 or SP6 polymerase promoters.Alternatively, antisense cDNA constructs that synthesize antisense RNAconstitutively or inducibly, depending on the promoter used, can beintroduced stably into cell lines.

Various modifications to the DNA molecules may be introduced as a meansof increasing intracellular stability and half-life. Possiblemodifications include but are not limited to the addition of flankingsequences of ribo- or deoxy-nucleotides to the 5′ and/or 3′ ends of themolecule or the use of phosphorothioate or 2′ O-methyl rather thanphosphodiesterase linkages within the oligodeoxyribonucleotide backbone.

5.3.2. Triplex DNA Formation

Oligodeoxyribonucleotides can form sequence-specific triple helices byhydrogen bonding to specific complementary sequences in duplexed DNA.Interest in triple helices has focused on the potential biological andtherapeutic applications of these structures. Formation of specifictriple helices may selectively inhibit the replication and/or geneexpression of targeted genes by prohibiting the specific binding offunctional trans-acting factors.

Oligonucleotides to be used in triplex helix formation should be singlestranded and composed of deoxynucleotides. The base composition of theseoligonucleotides must be designed to promote triple helix formation viaHoogsteen base pairing rules, which generally require sizeable stretchesof either purines or pyrimidines to be present on one strand of aduplex. Oligonucleotide sequences may be pyrimidine-based, which willresult in TAT and CGC triplets across the three associated strands ofthe resulting triple helix. The pyrimidine-rich oligonucleotides providebase complementarity to a purine-rich region of a single strand of theduplex in a parallel orientation to that strand. In addition,oligonucleotides may be chosen that are purine-rich, for example,containing a stretch of G residues. These oligonucleotides will form atriple helix with a DNA duplex that is rich in GC pairs, in which themajority of the purine residues are located on a single strand of thetargeted duplex, resulting in GGC triplets across the three strands inthe triplex. Alternatively, the potential sequences that can be targetedfor triple helix formation may be increased by creating a so called“switchback” oligonucleotide. Switchback oligonucleotides aresynthesized in an alternating 5′-3′,3′-5′ manner, such that they basepair with first one strand of a duplex and then the other, eliminatingthe necessity for a sizeable stretch of either purines or pyrimidines tobe present on one strand of a duplex.

5.3.3. Administration of Oligonucleotides

For anti-MIF therapeutic uses, the inhibitory oligonucleotides may beformulated and administered through a variety of means, includingsystemic, and localized, or topical, administration. Techniques forformulation and administration may be found in “Remington'sPharmaceutical Sciences,” Mack Publishing Co., Easton, Pa., latestedition. The mode of administration may be selected to maximize deliveryto a desired target organ in the body. For example, ¹²⁵I-MIF bindingstudies detailed in Section 12, infra, indicate that MIF ispreferentially distributed to the liver and kidney. Therefore,oligonucleotides designed to inhibit expression of the MIF-receptor maybe formulated for targeting to these organs; in this regard,liposome-encapsulated oligonucleotides may prove beneficial.Alternatively, MIF itself is produced in T cells and macrophages and, inresponse to endotoxin induction, in the pituitary. Therefore,oligonucleotides designed to inhibit the expression of MIF should beformulated for targeting to these organs.

For systemic administration, injection is preferred, includingintramuscular, intravenous, intraperitoneal, and subcutaneous. Forinjection, the oligonucleotides of interest are formulated in aqueoussolutions, preferably in physiologically compatible buffers such asHanks's solution, Ringer's solution, or physiological saline buffer. Inaddition, the oligonucleotides may be formulated in solid or lyophilizedform, then redissolved or suspended immediately prior to use. Systemicadministration may also be accomplished by transmucosal, transdermal, ororal means. For transmucosal or transdermal administration, penetrantsappropriate to the barrier to be permeated are used in the formulation.Such penetrants are generally known in the art. Transmucosaladministration may be through nasal sprays or suppositories. For oraladministration, oligonucleotides may be formulated into capsules,tablets, and tonics. For topical administration, the oligonucleotides ofthe invention are formulated into ointments, salves, gels, or creams, asis generally known in the art.

Alternatively, the oligonucleotides of the invention may first beencapsulated into liposomes, then administered as described above.Liposomes are spherical lipid bilayers with aqueous interiors. Allmolecules that are present in an aqueous solution at the time ofliposome formation (in this case, oligonucleotides) are incorporatedinto this aqueous interior. The liposomal contents are both protectedfrom the external microenvironment and, because liposomes fuse with cellmembranes, are efficiently delivered into the cell cytoplasm, obviatingthe need to neutralize the oligonucleotides' negative charge.

The introduction of oligonucleotides into organisms and cells for suchpurposes may be accomplished by several means. For mammalianadministration, each of the techniques described above for therapeuticoligonucleotide purposes may be used. In addition, other standardtechniques for introduction of nucleic acids into cells, including, butnot limited to, electroporation, microinjection, and calcium phosphateprecipitation techniques may be utilized.

5.4. Transgenic Animal Models

Transgenic animals may be engineered, using techniques well known tothose of skill in the art, whose cells may contain modified and/oradditional MIF and/or MIF receptor genes within their genomes. Forexample, animals may be produced that contain inactive MIF and/or MIFreceptor genes, or alternatively, may contain additional MIF and/or MIFreceptor genes.

Such transgenic animals may be used as model systems for the evaluationof cytokine responses in vivo, and may additionally serve as a means bywhich new drugs for the treatment of conditions involvingcytokine-mediated toxicity are identified and tested.

5.4.1. MIF and/or MIF Receptor Transgenes

DNA containing the nucleotide coding sequence for all or any portion ofthe MIF gene may be used to produce transgenic animals. Alternatively,all or any portion of a gene encoding a MIF receptor may be used.Further, insertions, substitutions, and/or deletions of one or morenucleotides of the MIF and/or MIF receptor genes may also be utilized inthe construction of the transgenic animals. Due to the degeneracy of thegenetic code, other DNA sequences which encode substantially the sameMIF or MIF receptor protein or a functional equivalent can also be used.The nucleotide coding sequence used to produce the transgenic animals ofthe invention may be regulated by any known promoter regulatorynucleotide sequence. If it is required that expression of the transgenebe limited to one or more specific tissues, tissue-specific enhancerregulatory sequences may also be used. Multiple copies of the genes orgene constructs may be stably integrated into the transgenic founderanimals.

Any of the nucleotide coding sequences and/or regulatory sequences thatwill yield any of the variants described here can be produced usingrecombinant DNA and cloning methods which are well known to those ofskill in the art. In order to produce the gene constructs used in theinvention, recombinant DNA and cloning methods which are well known tothose skilled in the art may be utilized (see Sambrook et al., 1989,Molecular Cloning, A Laboratory Manual, 2nd Ed., Cold Spring HarborLaboratory Press, NY). In this regard, appropriate MIF and/or MIFreceptor coding sequences may be generated from MIF cDNA or genomicclones using restriction enzyme sites that are conveniently located atthe relevant positions within the sequences. Alternatively, or inconjunction with the method above, site directed mutagenesis techniquesinvolving, for example, either the use of vectors such as M13 orphagemids, which are capable of producing single stranded circular DNAmolecules, in conjunction with synthetic oligonucleotides and specificstrains of Escherichia coli (E. coli) (Kunkel, T. A. et al., 1987, Meth.Enzymol. 154:367-382) or the use of synthetic oligonucleotides and PCR(polymerase chain reaction) (Ho et al., 1989, Gene 77:51-59; Kamman, M.et al., 1989, Nucl. Acids Res. 17: 5404) may be utilized to generate thenecessary nucleotide coding sequences. Appropriate MIF and/or MIFreceptor sequences may then be isolated, cloned, and used directly toproduce transgenic animals. The sequences may also be used to engineerthe chimeric gene constructs that utilize regulatory sequences otherthan the MIF and/or MIF receptor promoter, again using the techniquesdescribed here. These chimeric gene constructs would then also be usedin the production of transgenic animals.

Transgenic animals may also be produced in which the function of theendogenous MIF and/or MIF receptor genes has been disrupted. Toaccomplish these endogenous gene disruptions, the technique ofsitedirected inactivation via gene targeting (Thomas, K. R. andCapecchi, M. R., 1987, Cell 51:503-512) may be used. Briefly, vectorscontaining some nucleotide sequences homologous to the endogenous geneof interest are designed for the purpose of integrating, via homologousrecombination with chromosomal sequences, into and disrupting thefunction of, the nucleotide sequence of said endogenous gene.

5.4.2. Production of Transgenic Animal

Animals of any species, including but not limited to mice, rats,rabbits, guinea pigs, pigs, mini-pigs, and non-human primates, e.g.,baboons, squirrel monkeys and chimpanzees may be used to generate thetransgenic animals of the invention. Any technique known in the art maybe used to introduce the transgene into animals to produce the founderlines of transgenic animals. Such techniques include, but are notlimited to pronuclear microinjection (Hoppe, P. C. and Wagner, T. E.,1989, U.S. Pat. No. 4,873,191); retrovirus-mediated gene transfer intogerm lines (Van der Putten et al., 1985, Proc. Natl. Acad. Sci., USA82:6148-6152); gene targeting in embryonic stem cells (Thompson et al.,1989, Cell 56:313-321); electroporation of embryos (Lo, 1983, Mol. Cell.Biol. 3:1803-1814); and sperm-mediated gene transfer (Lavitrano et al.,1989, Cell 57:717-723); etc. (For a review of such techniques, seeGordon, 1989, Transgenic Animals, Intl. Rev. Cytol. 115:171-229, whichis incorporated by reference herein in its entirety.)

Once the founder animals are produced, they may be bred, inbred,outbred, or crossbred to produce colonies of the particular genotypedesired. Examples of such breeding strategies include but are notlimited to: outbreeding of founder animals with more than oneintegration site in order to establish separate lines; inbreeding ofseparate lines in order to produce compound transgenics that express thetransgene at higher levels because of the effects of additive expressionof each transgene; crossing of heterozygous transgenic mice to producemice homozygous for a given integration site in order to both augmentexpression and eliminate the need for screening of animals by DNAanalysis; crossing of separate homozygous lines to produce compoundheterozygous or homozygous lines; breeding animals to different inbredgenetic backgrounds so as to examine effects of modifying alleles onexpression of the transgene.

The present invention provides for transgenic animals that carry thetransgene in all their cells, as well as animals which carry thetransgene in some, but not all their cells, i.e., mosaic animals. Thetransgene may be integrated as a single transgene or in concatamers,e.g., head-to-head tandems or head-to-tail tandems.

5.5. Diagnostic Applications

MIF protein and/or mRNA levels may be monitored in an individual, usingstandard techniques, as an indication of the deleterious aspects of adisease condition. For the measurement of MIF protein concentrations,such monitoring techniques include, but are not limited to immunologicalassays such as, for example, Western blots, immunoassays such asradioimmuno-precipitation, enzyme-linked immunoassays, and the like. Forthe measurement of MIF mRNA concentrations, such techniques may include,for example, hybridization techniques such as Northern blot analysis, orany RNA amplification techniques, which may involve, for example,polymerase chain reaction (the experimental embodiment set forth inMullis, K. B., 1987, U.S. Pat. No. 4,683,202) ligase chain reaction(Barany, F., 1991, Proc. Natl. Acad. Sci. USA 88: 189-193)self-sustained sequence replication (Guatelli, J. C. et al., 1990, Proc.Natl. Acad. Sci. USA 87: 1874-1878), transcriptional amplificationsystem (Kwoh, D. Y. et al., 1989, Proc. Natl. Acad. Sci. USA 86:173-1177), or Q-Beta Replicase (Lizardi, P. M. et al., 1988,Bio/Technology 6: 1197).

6. EXAMPLE Molecular Cloning of MIF and Structural Comparison BetweenMurine and Human MIF

6.1. Materials and Methods

6.1.1 Materials

Reagents for polymerase chain reaction (PCR), reverse transcription(RT), and other molecular biology techniques were purchased from GibcoBRL (Grand Island, N.Y.) unless stated otherwise. PCR buffer was fromPerkin Elmer Cetus (Norwalk, Conn.); RNAse inhibitor “rRNasin” wasobtained from promega (Madison, Wis.); RNAzol™ B was from TEL-TEST, INC.(Friendswood, Tex.); and oligonucleotides were purchased from OLIGOSETC., INC. (Wilsonville, Oreg.). Manual DNA sequencing was performedwith the SEQUENASE® 2.0 system (United States Biochemical, Cleveland,Ohio, Tabor & Richardson, 1987). For automated DNA sequencing, the TagDyeDeoxy™ Terminator Cycle Sequencing Kit (Applied Biosystems Inc.,Foster City, Calif.) was utilized. Western blotting was performedfollowing a modification of the method by Anderson et al. (1982,Electrophoresis 3:135). Sodium dodecylsulfatepolyacrylamide gelelectrophoresis (SDS-PAGE) and Western blotting reagents were fromPierce (Rockford, Ill.). Polyclonal anti-rmuMIF antiserum was preparedfrom rabbits immunized with purified rmuMIF. Thioglycollate broth(Difco, Detroit, Mich.) was prepared according to the manufacturer'srecommendation, autoclaved, and stored protected from light at roomtemperature. E. coli 0111:B4 LPS and polymyxin B were purchased fromSigma (St. Louis, Mo.). LPS was resuspended in pyrogen-free water,vortexed, sonicated, aliquoted (5 mg/ml), and stored at −20° C. Serialdilutions of LPS were prepared in pyrogen-free water and sonicated(Branson 3210, Danbury, Conn.) for 10 min prior to use.

6.1.2. Molecular Cloning of Murine and Human MIF

Murine MIF was cloned from the mouse anterior pituitary cell lineAtT-20/D16v-F2 (American Type Culture Collection, Rockville, Md.). Cellswere plated at 1×10⁶ cells/ml in DMEM containing 50 μg/ml gentamicin(Gibco BRL), and 10% heat-inactivated fetal bovine serum (FBS) (HyClone,Logan, Utah). After 3 h of incubation at 37° C. in a humidifiedatmosphere with 5% CO₂, non-adherent cells were removed and theremaining adherent cells washed twice with DMEM/10% FBS. LPS (50 μg/ml)then was added and the cells were incubated for 16 h. At the end of thistime, cell culture medium was removed and total RNA was isolated withRNAzol™B according to the manufacturer's instructions. One μg of RNA wasreverse transcribed using oligo (dT)₁₂₋₁₈ and M-MLV reversetranscriptase in a 50 μL reaction. Five μL of cDNA was amplified by PCR(32 cycles; 1 min at 94° C., 1 min at 55° C., 1 min at 72° C.) using MIFprimers. A single DNA amplification product of expected size wasobtained and purified using the GENE CLEAN II® Kit (BIO 101 Inc., LaJolla, Calif.). The cDNA then was cloned into the plasmid pT7Blue andtransformed into Nova Blue competent E. coli using the pT7Blue T-VectorKit (Novagen, Madison, Wis.), Recombinant DNA was prepared from multiplewhite colonies using the Plasmid Magic™ Minipreps DNA PurificationSystem (promega) and sequenced manually in a Sequi-Gen II SequencingCell (BIO-RAD, Hercules, Calif.).

For human MIF cloning, Jurkat H33HJ-JA1 cells (American Type CultureCollection) were plated at 1×10⁶ cells/ml in RPMI containing 50 μg/mlgentamicin and 10% heat-inactivated FBS. After 3 h of incubation at 37°C. in a humidified atmosphere with 5% CO₂, non-adherent cells wereremoved and the remaining adherent cells washed twice with RPMI/10% FBS.Cells were incubated for another hour and the total RNA was isolated andsubjected to RT as described above. NdeI/MIF- and BamHI/MIF-fusionprimers (5′CCATATGCCGATGTTCATCGTAAACAC-3′ [SEQ ID NO:8] and3′CGGATCCTGCGGCTCTTAGGCGAAGG-5′ [SEQ ID NO:9]) were designed from ahuMIF cDNA sequence (Weiser et al., 1989, Proc. Natl. 30 Acad. Sci. USA86: 7522) and used to amplify human MIF cDNA as described above. Asingle PCR product of predicted size was obtained, purified using theGENE CLEAN II® Kit, and ligated into the NdeI/BamHI-digested pET11bvector (Novagen). E. coli DH5a was transformed with the ligation mixtureand the single recombinant colonies isolated after overnight growth.Plasmid DNA was prepared from eight clones and the MIF insert sequencedbi-directionally by automated methods using an ABI Model 373A DNAsequencer (Applied Biosystems Inc.).

6.1.3. Expression and Purification of Recombinant MIF

The recombinant pT7Blue clone containing muMIF cDNA was digested withthe restriction enzymes NdeI and BamHI, and the MIF insert isolated andligated into the NdeI/BamnHI-digested pET11b vector (Novagen). E. coliDH5a was transformed and the single recombinant colonies isolated andstored in 20% glycerol at −80° C. until use. Murine or humanMIF-containing pET11b plasmid DNA then was prepared and used totransform the E. coli BL21(DE3) expression strain (Novagen). One-litercultures were grown at 37° C. until the optical density at 600 nmreached 0.6-0.8. Isopropyl 1-thio-β-D-galactopyranoside (IPTG) was addedto a final concentration of 1 mM and the incubation continued at 37° C.for an additional 3 h. Bacteria then were harvested by centrifugationand the cell pellets frozen at −20° C. until use.

For protein purification, the bacterial pellets (corresponding to 50 mlof culture) were thawed and resuspended in 3.5 ml of Tris-bufferedsaline (50 mM Tris-HCl, 150 mM NaCl, pH 7.5). The bacteria were lysed byadding an equal volume of washed glass beads (106 microns; Sigma,G-8893) and vortexing the mixture vigorously for 10 min. Lysis wasconfirmed by examination under phase contrast microscopy. Glass beadswere removed by centrifugation at 1000 g for 10 min and the bacterialextract then was centrifuged at 38000 g for 30 min. The supernatant,representing the cleared bacterial lysate, was sterile-filtered througha 0.45 μm and then a 0.22 μm membrane filter and subjected to MONO Qanion exchange chromatography using a Fast Protein Liquid Chromatographysystem (FPLC) (Pharmacia, Piscataway, N.J.). The Mono Q column wasequilibrated with Tris-buffered saline. MIF was eluted with the samebuffer in the first flow-through fractions (3 ml), which were pooled andplaced on ice immediately. The MIF-containing fractions then wereapplied to a C8-SepPak reverse-phase (RP) column (Waters, Division ofMILLIPORE, Milford, Mass.) that had been washed first with methanol,followed by water. Unbound material was eluted by washing the resin with10 column volumes of water and 20% acetonitrile/water, respectively. MIFthen was eluted with 6 column volumes of 60% acetonitrile/water, frozenat −80° C., lyophilized, and kept at −20° C. until use. Forrenaturation, MIF was dissolved at a concentration of 200-400 μg/ml in20 mM sodium phosphate buffer (pH 7.2) containing 8 M urea and 5 mM DTT,and dialyzed against 20 mM sodium phosphate buffer (pH 7.2) containing 5mM DTT, followed by 20 mM sodium phosphate buffer (pH 7.2) alone.Renatured MIF was sterile-filtered and kept at 4° C. until use. The LPScontent of MIF preparations was determined by the chromogenic Limulusamoebocyte assay (Bio-Whittaker Inc., Walkersville, Md.). Attempts topurify rmuMIF from the cleared bacterial lysate by affinitychromatography with S-hexyl-glutathione-agarose beads (Sigma, H-7011)were performed following the method for single-step purification ofprotein/glutathione-S-transferase fusion constructs (Smith & Johnson,1988, Gene 67: 31).

6.1.4. Purification of Native MIF

Two grams of mouse liver acetone powder (Sigma, L-8254) were resuspendedin 20 ml of Tris-buffered saline, vortexed, and the insoluble materialremoved by centrifugation at 1000 g for 10 min. The supernatant,containing MIF and other hepatic proteins, was filtered (0.45 μmfollowed by 0.22 μm filter) and subjected to MONO Q/FPLC anion exchangechromatography as described above. MIF eluted with the firstflow-through fractions (3 ml), which were pooled and applied to a Pro Scation exchange column (BIO-RAD) that was equilibrated withTris-buffered saline. MIF eluted with Tris-buffered saline and theMIF-containing fractions again were recovered from the flow-through. TheMIF fractions then were pooled and applied to a C8-SepPak cartridge. MIFwas eluted with 60% acetonitrile/water, lyophilized, and stored asdescribed above. Attempts to purify liver MIF from mouse liversupernatant by affinity chromatography with S-hexyl-glutathione-agarosebeads were performed as described above.

6.1.5. Biochemical Characterization of MIF

SDS-polyacrylamide gel electrophoresis (SDS-PAGE) was performed in 18%gels under reducing conditions (Laemmli, 1970, Nature 227: 680). Thegels were either stained with silver or processed further for Westernblotting. For silver staining, gels first were fixed for 16 h in 50%methanol/10% acetic acid and then analyzed as described (Poehling &Neuhoff, 1981, Electrophoresis 2: 141). For Western blotting, proteinswere transferred to nitrocellulose membrane (Schleicher & Schuell,Keene, N.H.) by electroblotting at 50 V and 150 mA for 16 h usingCAPS-transfer buffer (10 mM CAPS, 20% methanol, pH 11.0). Membranes thenwere incubated with blocking buffer (50 mM Tris-HCl, 500 mM NaCl, pH7.5, 5% non-fat dry milk, 0.05% Tween-20) followed by incubation with a1:1000 dilution of polyclonal rabbit anti-rmuMIF antiserum in bindingbuffer (50 mM Tris-HCl, 500 mM NaCl, pH 7.5, 1% BSA, 0.05% Tween-20).After extensive washing in binding buffer, membranes were incubated witha 1:1000 dilution of horseradish peroxidase-conjugated goat anti-rabbitantibody (Pierce) in binding buffer, washed twice with binding buffer,twice with 50 mM Tris-Base (pH 7.5) containing 150 mM NaCl, anddeveloped with chloronaphthol/H₂0₂ substrate. Prestained proteinmolecular weight (M_(r)) markers ranged from 2.5-43 kDa (Gibco BRL).

N-terminal MIF sequence was determined by Edman degradation (Allen; 1981In: Sequencing of Proteins and Peptides, Elsevier, Amsterdam, N.Y.) andautomated gas-phase sequencing following a manufacturer's protocol(Applied Biosystems).

For N-glycosylation analysis, 0.1 μg of purified liver-derived MIF wasincubated with the endoglycosidase PNGase F according to themanufacturer's instructions (New England BioLabs, Beverly, Mass.).Samples then were mixed 1:1 with Laemmli electrophoresis buffer, boiledfor 5 min, and ¼ of the incubation mixture analyzed by SDS-PAGE/Westernblotting as described above. To control for digestion efficiency, IgGlight and heavy chains were digested in parallel and resolved on thesame gel. Digestion was assessed to be complete after 3 h incubationwith 5000 units/ml of PNGase F.

For analytical size exclusion chromatography, 40 μg of purified rmuMIFor rhuMIF was dissolved in 200 μl 20 mM sodium phosphate buffer (pH 7.2)containing 7 M GdnHCl. The sample was separated over a Superose 12 HR10/30 column (Pharmacia), equilibrated with the same buffer, and elutedat a flow rate of 0.5 ml/min. Size markers were from BIORAD (1.35-670kDa) and were chromatographed under the same conditions.

Mass spectrometric analysis (MS) of MIF was performed by matrix-assistedlaser desorption ionization MS as described elsewhere (Hillenkamp &Karas, 1990, Meth. Enzymol. 193: 280) using a Shimadzu Kratos KompactMALDI 3 V3.0.2 machine (Hannover, Germany). Twenty individual spectrawere accumulated for each mass analysis. In addition, rmuMIF wasanalyzed by ion-spray mass spectroscopy (IS-MS) using an API III triplequadrupole mass spectrometer with an IonSpray™ interface (Sciex,Toronto).

Purified, renatured rmuMIF (20 μg/ml) and MIF containing bacteriallysates (estimated MIF content: 50 μg/ml) were analyzed photometricallyfor glutathione-S-transferase (GST) activity by the method of Fjellstedtet al (1973, J. Biol. Chem. 248: 3702) using1,2-epoxy-3-(p-nitrophenoxy)propane (EPNP) as a substrate and bovine GST(Sigma, G-8386) as a positive control (Blocki et al., 1992, Nature 360:269). In this assay system, bovine GST (400 μg/ml) was found to have anenzyme activity of 0.014 units/ml with respect to the substrate EPNP.

6.1.6. Bioactivity Profile

The macrophage/monocyte migration inhibitory activity of recombinant MIFwas analyzed by studying the migration of human peripheral bloodmonocytes in modified Boyden chambers (Boyden, 1962, J. Exp. Med. 115:453). Monocytes were isolated from heparinized venous blood of healthydonors and resuspended at a concentration of 2.5×10⁶ cells/ml in Gey'sbalanced salts solution (Gibco BRL) containing 2% fatty acidfree bovineserum albumin (Sigma) and 20 mM Hepes (pH7.2). MIF-containing (0.0001-10μg/ml) solutions or buffer controls were placed in the lower compartmentof the Boyden chamber, covered tightly by a polyvinylpyrrolidone-freepolycarbonate filter (5-μm pore size, no. 155845, Costar Corp.,Cambridge, Mass.) and monocytes (1×10⁵) added to the compartment abovethe filter. The chambers then were incubated for 3 h at 37° C. in ahumidified atmosphere with 5% CO₂. At the end of this time, the filterswere recovered and the cells fixed and stained with Giemsa reagent.Monocytes then were counted as described previously (Sherry et al.,1992, Proc. Natl. Acad. Sci. USA 89: 3511).

TNFα production was quantitated in MIF-conditioned RAW 264.7 macrophagesupernatants by L929 cell cytotoxicity as described previously (Wolpe etal., 1988, J. Exp. Med. 167: 570). RAW 264.7 macrophages wereresuspended in RPMI/10% FBS, plated at 1×10⁶ cells/ml, incubated for 3hours at 37° C. in a humidified atmosphere with 5% CO₂, and washed twicewith RPMI/1% FBS. Cells were incubated for 12-14 hours with variousdoses of MIF diluted in RPMI/1% FBS. At the end of each experiment, cellculture media were collected, centrifuged (10 mM at 800 g), andsupplemented with 1 mM PMSF. MIF-induced TNFα activity was measuredimmediately afterwards. Polyclonal rabbit anti-recombinant murine TNFαantiserum (50 μl/ml) blocked TNFα activity completely, and MIF did notcontribute to TNFα activity as recombinant murine TNFα (rTNFα) (5 pg/mlto 1 μg/ml) cytotoxicity remained unchanged when rmuMIF (10 pg/ml to 10μg/ml) or anti-rmuMIF antibody were added to L929 cells together withrTNFα.

Nitric oxide (NO) production was quantitated in, MIF-conditioned RAW264.7 and thioglycollate-elicited peritoneal exudate macrophagesupernatants by measuring nitrite and nitrate content with the Griessreagent. RAW 264.7 macrophages were prepared and conditioned asdescribed above. In some experiments, cells were incubated for 1 h withrecombinant murine interferon-γ (IFN-γ) (100 IU/ml)(Boehringer-Mannheim, Indianapolis, Ind.) prior to the addition of MIF.Thioglycollate-elicited peritoneal exudate macrophages were obtainedfrom BALB/c mice that were injected intra-peritoneally 3-4 dayspreviously with 2 ml of sterile thioglycollate broth. Cells wereharvested under strict aseptic conditions by lavage of the peritonealcavity with 5 ml of an ice-chilled 11.6% sucrose solution. Aftercentrifugation (10 min at 800 g), cells were resuspended in RPMI/10% FBSand plated at a density of 2×10⁶ cells/ml. After 3 h of incubation,non-adherent cells (i.e. PMNs, lymphocytes) were removed with RPMI/1%FBS and the remaining adherent cells conditioned with MIF as describedfor RAW 264.7 cells. Trace concentrations of contaminating LPS wereneutralized by incubating MIF (1 and 10 μg/ml in cell culture media)with polymyxin B at a concentration of 10 and 100 ng/ml, respectively,for 30 mM at room temperature under sterile conditions. The mixture wascleared by centrifugation and added to the prepared macrophage cultures.The polymyxin B concentration added was approximately a 1000-fold higherthan necessary to neutralize the contaminating LPS present. For antibodyneutralization of MIF activity, rmuMIF (1 and 10 μg/ml in cell culturemedia) was treated with 20 μL/ml of anti-rmuMIF or normal rabbit controlserum and added to the macrophages as described above.

6.1.7. Conformational and Structural Stability Analysis

CD spectra were recorded on an Aviv Associates Model 62DSspectropolarimeter. The spectra represent the average of three scansrecorded at 25° C. in the range between 190 nm and 250 nm and werecollected at 0.25 nm intervals, with a band width of 1.5 nm and a timeconstant of 1.0 sec. The quartz cells (1 and 10 mm) used in all CDmeasurements were washed with 30% HCl in ethanol, rinsed with water andmethanol, and dried before used. Protein concentrations were determinedfrom stock solutions prepared in 20 mM phosphate buffer (pH 7.2) usingthe Bio-Rad protein assay (BIO-RAD). This assay was found to agree withvalues obtained by quantitative amino acid analysis. Thirty min beforeCD analysis, proteins were diluted from the stock solutions to a finalconcentration of 10 μM in 20 mM phosphate buffer (pH 7.2), unless statedotherwise. CD spectra are presented as a plot of the mean molarellipticity per residue ([θ], deg cm² dmol⁻¹) versus the wavelength.

For unfolding experiments, MIF stock solutions dissolved in 20 mMphosphate buffer (pH 7.2) were mixed with increasing volumes of 8 MGdnHCl (molecular biology reagent grade, Sigma) prepared in 20 mMphosphate buffer (pH 7.2) so as to achieve a final protein concentrationof 10 μM and the indicated final GdnHCl concentrations. Samples thenwere equilibrated at room temperature for 30 min prior to the recordingof CD spectra.

6.2 Results

6.2.1. Cloning of Murine and Human MIF

N-terminal protein sequencing and cDNA cloning were used to identify themurine homolog of MIF among the proteins secreted by LPS-stimulatedanterior pituitary cells. MuMIF cDNA was prepared from the total RNA ofLPS-stimulated anterior pituitary cells (AtT-20) and amplified with MIFprimers. MIF cDNA then was cloned into the pT7Blue T-vector andsubjected to DNA sequence analysis. The muMIF cDNA sequence obtainedfrom 6 plasmid clones was compared to a previously published humanT-cell MIF cDNA (Weiser et al., 1989, Proc. Natl. Acad. Sci. USA 86:7522). Murine MIF cDNA (FIG. 3) [SEQ ID NO:3] was found to display a88.2% sequence homology to this huMIF sequence over a 348 nucleotideopen reading frame and to be identical in sequence with recentlyreported murine 3T3 fibroblast, and murine embryonic eye lens MIF cDNAs(Lanahan et al., 1992, Mol. Cell. Biol. 12: 3919; Wistow et al., 1993,Proc. Natl. Acad. Sci. USA 90: 1272).

HuMIF cDNA was prepared by RT/PCR of RNA isolated from resting JurkatH33HJ-JA1 T-cells. MIF cDNA was amplified with flanking primers bearingNdeI/BamHI restriction sites, thus enabling the subsequent cloning ofthe huMIF amplification product directly into a NdeI/BamHI-digestedpET11b prokaryotic expression plasmid. Human MIF cDNA sequence then wasobtained by sequencing both DNA strands of 8 independently derived E.coli clones. This Jurkat T-cell MIF sequence was found to be identicalto a recently reported human fetal lens MIF cDNA (Wistow et al., 1993,Proc. Natl. Acad. Sci. USA 90: 1272) and to a glycosylation inhibitionfactor (GIF) cDNA (Mikayama et al., 1993, Proc. Natl. Acad. Sci. USA 90:10056), but differed from the first reported human T-cell MIF sequenceobtained from the T-CEMB cell line (Weiser et al., 1989, Proc. Natl.Acad. Sci. USA 86: 7522) by a single base change at position 318. ThisG→A substitution produces a conservative Ser¹⁰⁶→Asn¹⁰⁶ change in thededuced amino acid sequence and renders the human protein even morehomologous with murine MIF. Murine pituitary MIF cDNA and human Jurkatcell cDNA exhibited 88.5% identity over a 348 nucleotide open readingframe and the amino acid sequences deduced for murine (AtT-20) and human(Jurkat) MIF were found to be 90% identical over the 115 amino acids(FIG. 4). No apparent N-terminal signal sequences were evident in eitherthe murine or the human proteins. MIF thus joins a growing list ofcytokines, such as interleukin-1 (IL-1) (Rubartelli et al., 1990, EMBOJ. 9:1503), basic fibroblast growth factor (bFGF) (Jackson et al., 1992,Proc. Natl. Acad. Sci. USA 89: 10691), and a secreted form ofcyclophilin (Sherry et al., 1992, Proc. Natl. Acad. Sci. USA 89: 3511),which are released from cells by non-classical protein secretionpathways. Two potential N-glycosylation sites were found at positions 75and 110 of the muMIF amino acid sequence and were at nearly identicalpositions in the human Jurkat MIF sequence (positions 73 and 110). Threecysteine residues (positions 57, 60, and 81) also were at identicalplaces in the murine and human MIF predicted amino acid sequences.

6.2.2. Expression and Purification of Recombinant MIF from E. Coli

Recombinant muMIF and huMIF were expressed in E. coli by cloning murineand human MIF cDNA into the IPTG-inducible pET expression plasmidsystem. Initial attempts to express murine MIF from the pET15b vector,which created an N-terminal oligo-histidine fusion protein and allowedfor facile purification of recombinant protein by ion metal affinityChromatography (IMAC), were unsuccessful because the protein wasresistant to the subsequent thrombin cleavage necessary to remove theoligo-histidine leader. Thus, recombinant muMIF was expressed and thenpurified by conventional means and the muMIF cDNA subcloned into theplasmid pET11b. This produced a construct which bore a three amino acid(Met-Asp-Ser) leader sequence attached to the N-terminus of MIF. HuMIFalso was expressed from the pET11b vector but was engineered by DNAamplification to begin at the second amino acid of the open readingframe, with the start methionine of MIF provided by the NdeI restrictionsite. The correct expression of MIF was verified by N-terminal aminoacid sequencing of gelpurified protein (10 amino acids for muMIF),SDS-PAGE, and Western blotting with anti-AtT-20 MIF antibody. ThepET11b-derived MIF was used for the subsequent purification of bioactivemuMIF and huMIF.

Recombinant murine or human MIF were found to constitute 40% of thetotal supernatant protein of E. coli lysates. Anion exchangechromatography at pH 7.5 resulted in approximately an 80% purificationof murine and human MIF. Subsequent application to a C8 reverse-phasecolumn yielded pure protein for both the murine and the humanrecombinant proteins, as verified by the appearance of single bands ofpredicted M_(r) (12.5 kDa) by SDS-PAGE/silver staining and by massspectrometric analysis, infra. This simple and rapid two-step procedurewas used to purify 1 mg of murine or human MIF per 50 ml of E. coliculture. After renaturation, 0.8-0.9 mg of soluble, bioactiverecombinant MIF was obtained per 50 ml culture. The overall yield of MIFprotein from total bacterial supernatant protein was estimated to be20%. This two-step purification method was elected for reasons ofsimplicity and rapidity and to minimize protein losses by non-specificprecipitation. Purification by C8 chromatography using a disposable, lowvolume push column also served to remove the LPS carried over from E.coli host cells. Recombinant MIF purified by these procedures containedno more than trace amounts of LPS (4-8 ng LPS per mg MIF).

6.2.3. Purification of Native MIF

To assess more precisely the biological activities of recombinant MIF,native MIF was also purified from mouse tissue. Pituitary cells yieldedinsufficient quantities of MIF protein for biochemical characterization,but Western blotting showed that the liver was an abundant source of MIFin vivo. MIF was purified from liver by a method similar to that usedfor recombinant MIF purification. Mono Q anion exchange chromatographyresulted in partial purification and increased the relative MIF contentby approximately 25-fold. Subsequent application to the C8 push columnafforded almost pure MIF. Upon SDSPAGE/silver staining analysis however,5 additional bands were detected that could not be removed by varyingthe elution conditions of the RP chromatography. A Pro S cation exchangechromatography step therefore was added prior to the C8 step to removethese contaminants. The purity and homogeneity of liver MIF wasestablished by SDS-PAGE/silver staining, Western blotting usinganti-rmuMIF antibody, and mass spectrometric analysis. Laser desorptionMS demonstrated a single protein species of M_(r) 12,555. The MIFcontent of mouse liver acetone powder was estimated to be less than 0.1%of total protein and 2 grams of mouse liver powder were used to purifyto homogeneity 50 μg native liver MIF.

6.2.4. Biochemical Characterization of MIF

The M_(r) for liver murine MIF, rmuMIF, and rhuMIF were each between12000 and 13000 as estimated by reducing SDS-PAGE. Two potentialN-linked glycosylation sites were detected in the predicted amino acidsequence of murine and human MIF and thus the possibility that nativemuMIF was posttranslationally glycosylated in vivo was investigated.Endoglycosidase F (PNGase F) digestion of purified liver MIF followed byreducing SDS-PAGE/Western blotting analysis showed no shift in theobserved M_(r), indicating the absence of significant N-glycosylation ofnative liver MIF.

Gel exclusion chromatography of GdnHCl-denatured recombinant MIF showeda M_(r) of 14,256 and 13,803 for the murine and human proteins,respectively, indicating that MIF eluted predominantly as a monomer.

Laser desorption MS of MIF was performed to further assess glycosylationas well as the presence of any other significant post-translationalmodifications of MIF. Native MIF obtained from mouse liver wasdetermined to have a M_(r) of 12,555 (predicted MH⁺ average mass ofoxidized muMIF=12,503). By comparison, rmuMIF (which bore a 3 amino acidN-terminal leader sequence) was found to have a M_(r) of 12,814(predicted MH+ average mass of oxidized rmuMIF=12,801). Ion-spray MS ofrmuMIF yielded a similar mass (M_(r)=12,804). Using laser desorption MS,rhuMIF was found to have a M_(r) of 12,521 (predicted MH⁺ average massof oxidized rhuMIF=12,475). Differences observed between theexperimental and theoretical, predicted masses were within the margin ofexperimental error (0, 1-1%) for these analyses. These data essentiallyruled out the presence of significant post-translational modificationsof either native or recombinant MIF protein.

A 12 kDa protein purified from rat liver bearing N-terminal homologywith huMIF has been reported to bind to glutathione affinity matrix andto exhibit glutathione-S-transferase (GST) activity (Blocki et al.,1992, Nature 360: 269). Neither liver MIF nor rmuMIF was shown tospecifically absorb to glutathione-modified matrix.

6.2.5. Bioactivity Profile

Purified rmuMIF displayed significant migration inhibitory activity whentested on human peripheral blood monocytes (FIG. 5). A bell-shaped doseresponse curve was observed with peak activity occurring at 0.1 μg/ml.The precise basis for diminished inhibitory activity at high MIFconcentrations is not known, but similar dose-response profiles havebeen observed for chemotactic cytokines (Sherry et al., 1992, Proc.Natl. Acad. Sci. USA 89: 3511).

Recombinant MIF was used to induce TNFα release by the murine RAW 264.7macrophage cell line. Both rmuMIF and rhuMIF added at concentrations of0.1-10 μg/ml caused the release of bioactive TNFα in the ng/ml range(Table II). Despite species-specific differences, rhuMIF at 1 μg/ml wasfound to be slightly more active than rmuMIF on murine macrophages. Atthe same concentration (1 μg/ml), the TNFα-inducing activity of MIFobtained from mouse liver was found to be higher than the activity ofrmuMIF. When tested at a concentration of 0.1 μg/ml however, theTNFα-inducing activity of mouse liver MIF was identical to rmuMIF.Similar findings were obtained when thioglycollate-elicited mouseperitoneal macrophages were used to study MIF-induced TNFα release.Overall, the activity of purified native MIF was within the rangeobserved for the recombinant proteins.

TABLE II MIF-INDUCED SECRETION OF TNFα BY MOUSE MACROPHAGES^(a)Secretion of TNFα (ng/ml) induced by: MIF (μg/ml) rmuMIF rhuMIF NativeMIF 10 ND 3.9 ± 2.8 ND 1 1.2 ± 0.1 3.4 ± 2.8 6.9 ± 3.4 0.1 0.2 ± 0.3 2.9± 2.4 0.2 ± 0.1 0.01 0 ND 0 ^(a)RAW 264.7 macrophages were prepared andincubated with MIF for 12-14 h as described in Section 6.1.6. supra.After the incubations, macrophage culture supernatants were removed, andanalyzed by L929 cell toxicity assay for TNFα activity. TNFαmeasurements were performed in duplicate and TNFα activity is expressedas the difference between the level produced by MIFstimulated cells andby non-stimulated control cells. Data are the mean ± SEM of at leastthree separate experiments. ND: not determined.

Native and recombinant MIF also induced NO production in macrophages(Table III). However, significant MIF-induced NO production was observedonly when macrophages were incubated with IFN-γ prior to stimulationwith MIF. In RAW 264.7 macrophages, the activity of rhuMIF at 1 μg/mlwas found to be approximately 4-fold higher than rmuMIF. Native MIFobtained from mouse liver also was more active in this assay thanrmuMIF. MuMIF stimulated NO production from IFN-γ-primed,thioglycollate-elicited peritoneal exudate macrophages at levels whichwere comparable with those observed in RAW 264.7 cells. However, liverMIF at 1 μg/ml was not found to be more active than rmuMIF on thismacrophage cell type.

The synthesis of NO by macrophages is induced by ≧10 ng/ml of LPS andthis effect is potentiated by IFN-γ priming (Ding et al., 1988, J.Immunol. 141: 2407). To exclude the possibility that the trace amountsof LPS present in recombinant MIF preparations contribute to NOproduction, MIF-induced NO release was tested after neutralization ofLPS with a large excess of polymyxin B. The production of NO was notreduced by polymyxin B treatment, arguing against a potentiating rolefor LPS in the stimulation of macrophages by MIF. In further controlstudies, the addition of neutralizing anti-rmuMIF antiserum (20 μl/ml)inhibited rmuMIF (1 and 10 μg/ml)-induced NO production, furtherconfirming the specificity of macrophage stimulation by recombinant MIF.

TABLE III NO PRODUCTION IN MOUSE MACROPHAGES INDUCED BY MIF, ALONE OR INCOMBINATION WITH IFN-γ^(a) Formation of Nitrite (μM) induced by: MIF(μg/ml) rmu MIF rhuMIF Native MIF −IFN-γ 10^(a) 0.2 ± 0.2 3.7 ± 2.1 ND 1^(a) 0.5 ± 0.5 1.5 ± 1.0 0  0.1^(a) 0.3 ± 0.3 0 0  0.01^(a) 0  ND 0+IFN-γ 10^(a) 12.3 ± 2.5  25.3 ± 7.5  ND  1^(a) 5.7 ± 1.3 20.4 ± 5.7 9.8 ± 1.3  0.1^(a) 1.7 ± 0.7 8.4 ± 2.6 3.1 ± 1.0  0.01^(a) 1.9 ± 1.2 ND0.5 ± 0.2  1^(b) 12.5 ND   3.5 ^(a)RAW 264.7 macrophages were preparedand incubated with MIF for 12-14 h. At the end of incubation, 300 μL ofculture supernatant was removed, mixed with 600 μL of Griess reagent,and the concentration of nitrite measured. Nitrite measurements wereperformed in duplicate and nitrite production is expressed as thedifference between the level produced by MIF-stimulated cells versusnon-stimulated, or IFN-γ-treated control cells. Data are the mean ± SEMof at least three separate experiments. ND: not determined. ^(b)NOproduction by MIF-stimulated thioglycollate-elicited peritonealmacrophages in place of RAW 264.7 macrophages. Data are the mean of twoseparate experiments.

6.2.6 Conformational and Structural Stability

Far-UV CD has been used widely to analyze the solution conformation ofproteins and to verify native and renatured protein structures(Greenfield & Fasman, 35 1969, Biochemistry 8: 4108). Far-UV CDspectroscopy and GdnHCl-induced unfolding studies were performed withrecombinant MIF to begin to assess the secondary structure and thestructural stability of this protein in a solution environment. Noconcentration dependent changes in the CD spectra of MIF were observedwithin the range studied (1.6-32 μM MIF). The spectrum for rmuMIF showeda pronounced positive ellipticity at 192 nm, a broad negativeellipticity between 209 nm and 222 nm, and small distinct minima at 210nm and 222 nm. These data are consistent with a highly ordered secondarystructure containing predominantly fl-sheet, but also a-helixconformation. The spectrum for rhuMIF showed a prominent positiveellipticity at 197 nm and a strong negative ellipticity between 211 nmand 225 nm was observed. This CD spectrum also was consistent with ahighly ordered secondary structure, but was suggestive of a higherβ-sheet content and a lower α-helix content than rmuMIF. Secondarystructure compositions for both proteins then were estimated fromcomputational fits of the CD spectra over the 190-250 nm range using themethod of Brahms & Brahms (1980, J. Mol. Biol. 138: 149), which isfrequently employed for the analysis of fi-sheet-rich proteins.Recombinant muMIF was estimated to contain 42.2% β-sheet, 15.3% α-helix,7.2% β-turn, and a remainder of 35.3% in random coil. Recombinant huMIFcontained a somewhat greater extent of ordered structure, with 72.9%β-sheet, 0% α-helix, 8.3% β-turn, and 17.3% random coil conformation.Secondary structure calculations following the methods of Chou & Fasman(1978, Adv. Enzymol. 47: 45) and Garnier et al. (1978, J. Mol. Biol.120: 97) also predicted that both muMIF and huMIF exhibit a highlyordered secondary structure. Chou/Fasman calculations predicted thefollowing secondary structure compositions—rmuMIF: 35.6%β-sheet, 10.6%α-helix, and 21.2% β-turn; rhuMIF: 26.1% β-sheet, 30.4% α-helix, and17.4% β-turn. Garnier/Osguthorpe/Robson analysis predicted the followingstructures—rmuMIF: 39%/3-sheet, 8.5% α-helix, and 20.3% β-turn; rhuMIF:24.4%β-sheet, 19.1% α-helix, and 25.2%/3-turn. The CD data, togetherwith the secondary structure predictions according to the Chou/Fasmanand Garnier/Osguthorpe/Robson calculations, indicate that bothrecombinant murine and human MIF contain a high fraction of β-sheetconformation (24.4%-72.9%). The spectroscopic data and primary sequencepredictions showed a similar a-helical content for rmuMIF (8.5%-15.3%).Although the secondary structures predicted by Chou/Fasman andGarnier/Osguthorpe/Robson calculations suggested that there was asignificant α-helical content in huMIF, the CD analysis did not show anydetectable a-helical elements in rhuMIF.

Helical conformations induced at the membrane-water interface have beensuggested to be important for ligand membrane interactions and toinfluence the binding of ligands to receptors (Kaiser & Kezdy, 1983,Proc. Natl. Acad. Sci. USA 80:1137; Fry et al., 1992, Biopolymers32:649; Erne et al., 1985, Biochemistry 24: 4263).2,2,2-Trifluoro-ethanol (TFE) induces and stabilizes the helicalconformation of proteins with a helix-forming propensity and has beenused to mimic the influence of membranes on polypeptide conformation(Sönnichsen et al., 1992, Biochemistry 31: 8790). To begin to testwhether the α-helical content of either murine or human MIF might beincreased in the membranous environment, far-UV CD analysis of rmuMIFand rhuMIF was performed in 50% TFE. TFE significantly increased theα-helical content of rmuMIF, as demonstrated by a larger positiveellipticity at 192 nm and pronounced negative ellipticities at 208 nmand 222 nm. The calculated fraction of α-helix conformation increasedfrom 15.3% to 34.7% in the presence of TFE. Recombinant huMIF, which wasfound to have no measurable α-helical elements in aqueous solution,showed a significant content of α-helix in the presence of TFE asevident by a shift of the positive ellipticity from 197 nm to 192 nm anddistinct negative ellipticities at 208 nm and 222 nm. Computationalanalysis of the spectroscopic data showed that the percentage of α-helixincreased from 0% to 36.3%. These data indicate that both murine andhuman MIF are likely to adopt significant α-helical conformation in amembranous environment.

Protein structural stability can be quantitated by determining the freeenergy of unfolding, ΔG_(N-U), where N is the fraction of protein in thenative state and U is the fraction in the unfolded state. One methodused frequently to assess protein stability is to measure protein meanmolar ellipticity per residue as a function of wavelength and GdnHClconcentration (Pace, 1975, CRC Crit. Rev. Biochem. 3: 1). Unfoldingcurves expressed as the percentage of unfolded protein relative tonative protein (i.e. the change in ellipticity at 222 nm) over theconcentration of GdnHCl provide two measures of structural stability: 1)the midpoint of unfolding, [GdnHCl]_(0.5), which can be deduced from thelinear part of the unfolding curve and 2) the free energy of unfoldingat zero denaturant concentration, ΔG°_(N-U), which can be extrapolatedfrom the unfolding curve by the linear extrapolation method (LEM)(Santoro & Bolen, 1992, Biochem. 31: 4901). Both measures are based onthe premise that unfolding follows a reversible two-state mechanism andthat unfolding free energy is linearly dependent on denaturantconcentration (Greene & Pace, 1974, J. Biol. Chem. 249: 5388).

The CD spectra of rmuMIF were recorded between the wavelengths 210nm-250 nm in the presence of increasing concentrations of GdnHCl (0-7M). Recording below 210 nm was not performed due to strong scatteringeffects at these wavelengths. The MIF spectrum, defined in the absenceof GdnHCl, changed visibly when ≧1.5 M GdnHCl was present, indicating asignificant loss of conformational integrity. The featureless spectrumfor fully denatured MIF in the presence of 7 M GdnHCl was similar tospectra observed for other fully unfolded proteins (Greenfield & Fasman,1969, Biochem. 8: 4108). For quantitation of GdnHCl-induced unfolding,the unfolding of MIF α-helical structures was plotted by expressing therelative change in [θ] at 222 nm (percent unfolded) with respect toincreasing concentrations of GdnHCl. The midpoint of unfolding wasobserved to be at 1.8 M GdnHCl. Next, the LEM was applied to extrapolateΔG°_(N-U), from a plot of ΔG_(N-U), versus the concentration ofGdnHCl([GdnHCl]). Data points of the linear portion of the curve couldbe replotted according to the following equation:

ΔG_(N-U)=ΔG°_(N-U) −m[GdnHCl]

where m is the slope of the curve, and subjected to linear regressionanalysis. Murine MIF thus was calculated to have a ΔG°_(N-U) of 11.75kJ/mol. By comparison, globular proteins generally exhibit ΔG°_(N-U)values of approximately 50±15 kJ/mol (Pace 1990, Trends Biochem. Sci.15: 14).

7. EXAMPLE In Vivo Administration of MIF Increases Endotoxin-InducedLethality and Anti-MIF Treatment Confers Protection

The following experiment was initially carried out to test the efficacyof purified MIF as a protective factor against endotoxin-inducedlethality in animals. Surprisingly, the in vivo administration of MIFpotentiated LPS lethality in mice and exacerbated the response toendotoxin challenge. Furthermore, mice pretreated with an anti-MIFantiserum were significantly protected against endotoxin-inducedlethality. Anti-MIF treatment also protected against TNF α-inducedlethality.

7.1. Materials and Methods

7.1.1. Mice

Nine-week-old BALB/c mice (19-21 g of weight) were utilized for thisstudy. A total of 20 animals in each category (i.e., LPS, LPS plus MIF,anti-MIF, preimmune serum and saline-treated) were included in theexperiments described below.

7.1.2. In Vivo Treatment with Endotoxin and MIF

Mice were injected i.p. with 15 mg/kg E. coli 0111:B4 LPS(lipopolysaccharide) in saline. MIF prepared as described in Section 6,supra, was administered i.p. to mice at 5 mg/kg in saline at 0 and 12hours after LPS treatment. Both control and treated animal groups weremaintained under the standardized conditions of the animal facilitycolony rooms and monitored for survival.

7.1.3. Anti-MIF Treatment

Anti-MIF antiserum was produced by immunization of rabbits with 100 μgof recombinant murine MIF purified from E. coli transfected with thecoding sequence in an expression vector. Anti-MIF serum and normalrabbit serum (NRS) showed no cross-reactivity with E. coli 0111:84 LPSas assessed by Western blot analysis and LPS-specific ELISA.

Mice were pretreated with anti-MIF antiserum (containing <0.2 ngLPS/ml), preimmune NRS (containing <3 ng LPS/ml), or a saline control 24hours and 2 hours prior to challenge with LPS (i.p. at 17.5 mg/ml, anamount of E. coli 011.B LPS that had been established as an LD₇₅ forsaline-injected control mice in prior dose-ranging studies).Pretreatments consisted of 200 μl of sera or 200 μl 0.9% (w/v) NaCl ascontrol, administered i.p.

TNFα challenge consisted of 0.7 μg in Tris-buffered saline administeredi.p. concurrently with 18 mg D-galactosamine.

7.2. Results

The effects of MIF on endotoxin-induced lethality were tested bycomparing the survival curves of animals challenged in the presence andabsence of administered exogenous recombinant MIF. Briefly, each animalwas injected i.p. with 300 μg LPS. Half of the animals were additionallyadministered 100 μg MIF i.p. at 0 and 12 hours post-LPS challenge.

The pooled data representing the results of two such experiments aredepicted in FIG. 6A in graphic form. These experiments demonstrate thatMIF potentiated LPS lethality in the mice tested. First, theLPS/MIF-treated mice showed an accentuated rate of mortality in that adifferential effect of MIF administration, relative to that of theLPS-only mice, was seen beginning approximately 20 hours post LPSchallenge. Second, the overall survival of animals in the two categoriesdemonstrated the increased cumulative mortality of animals exposed toMIF in addition to LPS, with only 15% of the LPS/MIF mice surviving at84 hours post-LPS challenge compared to 65% of the LPS-only treated micesurviving to this time point. The overall comparison in a two tailedFisher's exact test is P=0.003.

The effects of MIF on endotoxin- and TNFα-induced lethality were testedby comparing the survival of animals challenged after pretreatment withanti-MIF antisera to those animals pretreated with preimmune sera andsaline controls. Briefly, animals were injected, i.p., with eitheranti-MIF antisera, preimmune sera, or saline, 24 hours and 2 hours priorto challenge with either LPS or TNFα.

FIG. 6B illustrates the pooled data representing the results of twoexperiments in which the mice were challenged with LPS. As can be seen,mice pretreated with anti-MIF antisera were significantly protected fromLPS-induced lethality, with all mice surviving until the last recordedtimepoint, 86 hours post-challenge. In contrast, only 25% of the animalsthat had been pretreated with the saline control were still alive at 84hours post-LPS challenge, and only 50% of those pretreated withpreimmune sera were alive. A statistical analysis of these resultsindicated that the chances of these results occurring under the nullhypothesis were, for the overall comparison: P<0.00001; anti-MIF vs.saline: P<0.00001; anti-MIF vs. NRS: 0.0004; and NRS vs. saline: 0.19.

Table IV illustrates the pooled data representing the results of threeexperiments in which the mice were challenged with TNFα andgalactosamine. As recorded above in similar experiments with LPSchallenge, mice pretreated with anti-MIF antisera were significantlyprotected from TNFα-induced lethality, with all mice surviving until thelast recorded timepoint. In contrast, only 50% of the animals that hadbeen pretreated with the saline control were still alive at 120 hourspost-TNFα challenge, and only 60% of those pretreated with preimmunesera were alive. Any marginal benefits afforded by pretreatment withpreimmune control sera in these experiments most likely reflects thewell-known but small beneficial effect of exogenous gamma globulintreatment.

TABLE IV Days after TNFα and galactosamine challenge Treatment Group 1 23 4 5 Saline  9/10  9/10  9/10 5/10 5/10  90%  90%  90% 50% 50%Pre-immune 10/10 10/10 10/10 6/10 6/10 100% 100% 100% 60% 60% Anti-MIF10/10 10/10 10/10 10/10  10/10  100% 100% 100% 100%  100%  Fraction ofthe treatment groups surviving after TNFα + galactosamine treatment onday 0.

These experiments demonstrate that the immunological neutralization ofMIF had the ability to protect against both LPS-induced and TNFα-inducedlethality in the mice tested, as evidenced by the sparing by anti-MIFtreatment of animals that would otherwise be expected to die. Thus,these results not only prove that exogenously administered MIF has adeleterious effect on the shock process, but also demonstrate that theneutralization of endogenous MIF protects against endotoxin-induced andTNFα-induced lethality due to shock.

8. EXAMPLE Administration of Exotoxin Induces MIF Secretion and Anti-MIFTreatment Confers Protection

8.1. Materials and Methods

8.1.1. Cell Preparation and MIF Induction

RAW 264.7 murine macrophages were obtained from the American TypeCulture Collection (Rockville, Md.). Cells were grown in RPMI 1640medium (Gibco, Grand Island, N.Y.), 10% heat-inactivated fetal bovineserum (FBS) (HyClone, Logan, Utah), 50 μg/ml of carbenicillin andgentamicin. RAW 264.7 macrophages were washed with fresh medium,harvested by gentle scraping, resuspended in RPMI/10% FBS, and incubatedat 3×10⁶ cells/well in 3.5-cm tissue culture plates (Linbro®, Flow,McLean, Va.). After 3 h of incubation at 37° C. in a humidifiedatmosphere with 5% CO₂, nonadherent cells were removed and wells werewashed twice with RPMI/1% FBS. Cells then were incubated for 12-15 hwith TSST-1 (at concentrations ranging from 100 ng/ml to 100 attog/ml).At the end of experiment, cell-culture media were collected andcentrifuged (10 min at 800 g). Cell-conditioned media were concentrated10-fold by membrane filtration (10 kD cut-off) (Centricon-10, Amicon,Beverly, N.Y.).

Thioglycollate-elicited peritoneal macrophages were obtained from BALB/cmice that were injected i.p. 3-4 days previously with 2 ml of sterilethioglycollate broth. Cells were harvested under strict asepticconditions by lavage of the peritoneal cavity with 5 ml of anice-chilled 11.6% sucrose solution. After centrifugation (10 min at 800g), cells were resuspended in RPMI/10% FBS, enumerated and plated at adensity of 3−4×10⁶ cells/well. Cells were incubated for 12 to 15 h withTSST-1 (at concentrations ranging from 100 ng/ml to 100 attog/ml).

Murine T-cells (LBRM-33, a T lymphoma cell-line) were obtained from theAmerican Type Culture Collection (Rockville, Md.). Cells were grown inRPMI 1640 medium (Gibco, Grand Island, N.Y.), 10% heatinactivated fetalbovine serum (FBS) (HyClone, Logan, Utah), 0.05 nM 2-mercaptoethanol and50 μg/ml of gentamicin. Cells were washed with fresh medium, harvestedby gentle scraping, resuspended, and incubated at 1×10⁶ cells/well in3.5-cm tissue culture plates (Linbro®, Flow, McLean, Va.). After 3 h ofincubation at 37° C. in a humidified atmosphere with 5% CO₂, nonadherentcells were removed and wells were washed twice with RPMI/1% FBS. Cellsthen were incubated for 12-15 h with TSST-1 (at concentrations rangingfrom 100 ng/ml to 100 attog/ml). At the end of experiment, cell-culturemedia were collected and centrifuged (10 min at 800 g). Cell-conditionedmedia were concentrated 10-fold by membrane filtration (10 kD cut-off)(Centricon-10, Amicon, Beverly, N.Y.).

Samples were resolved on 18% SDS polyamilamide gels and analyzed byWestern blotting as described above.

8.1.2. Anti-MIF Treatment

Eight to 10-week-old (19-21 g) female BALB/c were purchased from CharlesRiver (Kingston, N.Y.). Animals were housed in groups of 5 or 10 miceper cage with free access to food and water and were acclimatized for 5days prior to experimentation. BALB/C mice were injected i.p. with 200μl of polyclonal rabbit anti-MIF serum or normal rabbit serum (controls)followed 2 hr later by a lethal i.p. injection combining 50 μg of TSST-1with 20 mg D-galactosamine. Mice were observed for 7 days.

8.2. Results

TSST-1 was found to be an extremely potent inducer of MIF secretion bymacrophages (both RAW 264.7 and peritoneal macrophages) and T cells invitro. Dose-response studies of TSST-1-induced MIF secretion showed twopeaks of MIF secretion. The first peak occurred at 10 μg/ml of TSST-1and the second at 100 attog/ml (=10⁻⁴ μg/ml). Anti-MIF treatmentmarkedly reduced mortality from 87.5% (controls) to 33% (p=0.05,two-tailed Fisher's exact test) (FIG. 7).

9. EXAMPLE MIF is a Primary Inflammatory Mediator in ParasiticInfections

9.1, Production of MIF in Malaria Infection

During malaria infection, hemozoin results from the pericyte-specificpolymerization of heme molecules liberated by the catabolism of redblood cell hemoglobin by intra-erythrocytic forms of Plasmodium, andthis “malaria pigment” accumulates within the reticuloendothelial systemof infected individuals. The structure of hemozoin is known, andhemozoin can be chemically synthesized by polymerizing ferric heme inacetic acid, and separated from unreacted heme by sequential extractionin NaHCO₃ buffer (pH 9.2) and ethanol. Endotoxin-free synthetichemozoin, prepared as above, was added to the murine monocyte cell lineRAW 264.7. The treated cells secreted a protein of 12.5 kDa which wasshown to be MIF by Western blotting and N-terminal sequence analysis.Thus, hemozoin was a potent inducer of MIF in vitro at as little as 1nmole heme equivalents. Furthermore, co-incubation of hemozoin withIFN-γ at 10-100 U/ml further increased MIF release.

To further assess the role of MIF in malaria infection, blood was drawnfrom C3H/HeN mice exhibiting a low level of chronic parasitemia withPlasmodium berghei (10% parasitized red cells). Five μl serum aliquotswere reacted with an anti-MIF antibody and analyzed by Western blotting.This relatively low level of parasitemia was associated with readilydetectable levels of circulating serum MIF. Levels of parasitemia in the10% range were sufficient to maintain persistent MIF production for aslong as 28 days.

In addition, 500 nmoles of endotoxin-free synthetic hemozoin wasinjected i.p. into C3H/HeN mice and blood sampled 24 and 48 hours later.The sera were separated and assayed for MIF by Western blotting and TNFαby L929 cytotoxicity assays. Although circulating TNFα levels wereundetectable in this study (<50 pg/ml), MIF was readily detected in seraat 24 and 48 hours. Hence, a low level parasitemia was associated withcirculating MIF in vivo. MIF appeared in sera under circumstances whenTNFα was not detectable. Hemozoin alone was found sufficient to induceMIF production in vitro and in vivo. Taken together, these results showthat MIF is an important early mediator in the host response to malariainfection. Furthermore, this observation indicates that anti-MIF therapymay be effective in ameliorating the pathological sequelae ofparasite-induced cytokine release.

10. EXAMPLE Anti-MIF Abrogates Antigen-Specific Immune Responses

This example demonstrates that anti-MIF antibody can substantiallyreduce the magnitude of an antigenspecific immune response. Thisindicates that MIF is a critical component of an immune response, andthus anti-MIF treatment may be useful in substantially reducing anundesired immune reaction such as autoimmunity and allergy.

10.1 Anti-MIF Inhibits Antigen-Specific T Cell Proliferation

Ten BALB/c mice were primed in vivo with RNase A emulsified in completeFreund's adjuvant at 1:1 ratio. Each mouse received 20 μg of RNase A in0.2 ml i.p. After 2-3 weeks, splenic T cells were isolated and theirproliferative activity assayed. In a microtiter plate, 4×10⁵ T cellswere incubated with 5×10⁵ antigen-presenting cells per well. While theaddition of RNase A led to an increased proliferation of the in vivoprimed T cells as measured by ³H-thymidine incorporation, thisproliferation was reduced to control levels when anti-MIF antibody wasalso added.

10.2. Anti-MIF Inhibits Antigen-Specific Inflammatory Response

BALB/c mice were primed in the left rear footpad and in the neck musclewith complete Freund's adjuvant at 0.2 ml per injection. After 10-14days, the mice were boosted in the right rear footpad with 50 μl ofpurified protein derivative (of tuburculin; PPD) with or without 500 ofanti-MIF antibody. When the width of the right rear footpads of theanimals were measured with calipers 48 hours later as an indication ofdelayed-type hypersensitivity reaction, anti-MIF treatment was shown toreduce the swelling in the footpads by greater than 50% as compared tocontrols. Analysis by light microscopy also revealed histopathologicalevidence of reduced inflammation in footpads of anti-MIF treated mice.

11. EXAMPLE Production of Migration Inhibitory Factor by the PituitaryGland

The following example describes the production of MIF by pituitary cellsin vitro and in vivo.

11.1. Materials and Methods

11.1.1. Cells

The murine anterior pituitary cell line AtT-20 was obtained fromAmerican Type Culture Collection and maintained in low serum medium.

11.1.2. Induction of MIF

AtT-20 cells were cultured for 24 hours in serumfree medium (enrichedserum-free Joklik's medium from Gibco) containing various concentrationsof LPS (E. coli LPS 0111:84 from Sigma) at 0.5 μg/ml-50 μg/ml. Two ml ofconditioned media was removed from 1.5×10⁵ cells and concentrated100-fold by membrane filtration (10K cut off) prior to SDSpolyacrylamide gel electrophoresis (18%). Silver staining analysis ofthe gels revealed a 12.5 kDa protein band. This protein was transferredto polyvinylidene difluoride membrane and its primary N-terminal aminoacid sequence was determined by automated gas-phase sequencing (AppliedBiosystems).

11.1.3. Western Blot

MIF release from pituitary (AtT-20) cells was analyzed by Westernblotting with anti-MIF antibody (FIG. 8). 1.5×10⁵ pituitary cells werecultured serum-free for 18 h with different concentrations of LPS. Aftertransfer, nitrocellulose membranes were incubated with rabbit anti-rMIFserum (1:1000 dilution) and bound antibody visualized with horseradishperoxidase-conjugated goat anti-rabbit antibody. 100 ng of rMIF waselectrophoresed on the same gel for reference. Control supernatants (C)contained 1 μg/ml LPS that was added after removal of cells. Pre-immuneserum showed no reactivity.

11.1.4. Immunocytochemistry

Cells were cultured for 16 h in the presence (FIG. 9B) or absence (FIG.9A) of 25 μg/ml LPS, washed, fixed with formaldehyde, and incubated withanti-rMIF serum (1:1000 dilution) prior to staining withimmunoperoxidase-linked goat anti-rabbit antibody. No staining wasobserved with pre-immune serum or with immune serum that had beenpre-incubated with rMIF.

11.1.5. Detection of MIF mRNA

Pituitary mRNA analyses from two representative mice are shown for eachtime interval (FIG. 10). RTpeR analysis utilized murine MIF primers (22cycles), β-actin primers (25 cycles), or CD2 primers (pituitary cDNA: 35cycles, control spleen cDNA: 25 cycles) as shown. Nine-week old micewere injected intraperitoneally with LPS at a dose of 2.25 mg/kg andsacrificed 0, 6, 16, 24 and 48 h later. Pituitary RNA was prepared byextraction with RNAzol (Biotecx Laboratories, Inc.) from a total of fourmice per time interval. After reverse transcription with oligo-dT, DNAamplification products were analyzed by agarose gel electrophoresis.Reverse transcription efficiency was assessed and normalized relative toβ-actin mRNA. The murine MIF primers were: 5′-CCATGCCTATGTTCATCGTG-3′[SEQ ID NO:10] and 3′-GA-ACAGCGGTGCAGGTAAGTG-5′ [SEQ ID NO:11] and weredesigned to amplify a 381 bp sequence that spanned at least one intron,as determined by Southern hybridization and internal restriction enzymeanalysis of mouse genomic DNA. Murine intron-spanning CD2 primers were5′CCTGGTCGCAGAGTTTAA-3′ [SEQ ID NO:12] and 3′-TCTGTTCCTTGCAAGACC-5′ [SEQID NO:13], and β-actin primers were 5′-GTGGGCCGCTCTAGGCACCA-3′ [SEQ IDNO:14] and 3′-TGGCCTTAGGGTTCAGGGGG-5′ [SEQ ID NO:15].

11.1.6. Polymerase Chain Reaction

RNA was analyzed (FIG. 11) at 0 hand 24 h after LPS injection (2.25mgjkg) by competitive PCR as described (Li et al., 1991, J. Exp. Med.174:1259-1262), utilizing a 273 bp competitive MIF template. Thecompetitive template was added in the range of 0.1 to 5 pg per reactionas shown in FIG. 11 and co-amplified with cDNA utilizing the MIF primersdescribed in Section 9.1.5., supra. At 0 h, equivalent amounts of cDNAand competitor DNA amplification products appear in the lane showing0.35 pg of MIF competitor template. At 24 h, equivalent amounts of cDNAand competitor DNA amplification products appear in the lane showing 1.0pg of MIF competitor template.

11.1.7. Expression of MIF Protein In Vivo

Pituitary MIF protein content was measured in BALB/c and C3H/HeJ micechallenged with LPS (FIG. 12). A similar LPS-dependent decrease inpituitary MIF protein content was observed in both C3H/HeN andBALB/c^(nu/nu) mice as in BALB/c mice. Mice (N=2-5 per time point) wereinjected intraperitoneally with LPS at 2.25 mg/kg, pituitary lysateswere prepared at intervals, and protein analyzed by Western blotting.Aliquots representing ¼ of a pituitary were electrophoresed, transferredto nitrocellulose membrane, and incubated with anti-rMIF serum (1:1000dilution). Bound antibody was visualized by incubation with horseradishperoxidase-conjugated goat anti-rabbit antibody and substrate.

Nine-week-old mice were injected intraperitoneally with LPS at 2.25mg/kg and serum samples (5 μl each) obtained at indicated time intervalswere analyzed by Western blotting. Single bands corresponding to MIFwere observed and quantified by laser densitometry (Shimadzu FDU-3,CS-9000 U). Recombinant MIF standards were electrophoresed andtransferred in adjacent lanes. Scanning integrals are presented asrelative MIF content normalized to the respective 0 h zone on eachmembrane. Each plotted point is the mean±SEM obtained for individualsera from multiple animals (N=2-5 per time point).

11.2. Results

The murine anterior pituitary cell line AtT-20 was cultured under lowserum conditions in the presence of increasing amounts of bacterialendotoxin. At time intervals, the conditioned media was collected,concentrated, and analyzed for the presence of secreted proteins by SDSpolyacrylamide gel electrophoresis. Endotoxin stimulation was observedto result in the specific time- and concentration-dependent secretion ofa 12.5 kDa protein. The specificity of the secretory response wasconfirmed by the absence of LPS-induced cytopathicity, as determined bythe lack of an LPS-related increase of supernatant lactate dehydrogenaseactivity. These measurements revealed no LPS-dependent increase inenzyme activity, despite prolonged (24 h) incubation under serum-freeconditions (0 μg LPS: 112.8±17.9 IU/L, 50 μg LPS: 119.7±13.1 IU/L;P=NS). The 12.5 kDa protein was isolated, subjected to N-terminalmicrosequencing, and identified as the murine homolog of human MIF (96%identity over 27 amino acids).

Cellular activation by LPS is known to be potentiated by serum factorssuch as LPS-binding proteins that interact with specific cell surfacereceptors (Schuman et al., 1990, Science 349:14291431; Wright et al.,1990, Science 249:1431-1433). Supplementation of culture medium with 1%fetal bovine serum increased markedly the secretion of MIF frompituitary cells stimulated by LPS. As little as 100 pg/ml of LPS wasfound to induce pituitary cell secretion of MIF (FIG. 8). In contrast,MIF was not released by incubation with the inflammatory mediators tumornecrosis factor α, (1-100 ng/ml), interleukin-1β (1-100 ng/ml),interleukin-6 (1-100 ng/ml), or interferon-γ (1-100 ng/ml). Westernblotting analysis of pituitary cell lysates also revealed that resting,non-stimulated cells contained significant amounts of pre-formed MIF.Immunocytochemistry studies confirmed these results and showed thedisappearance of substantial amounts of immunoreactive, intracellularMIF within 16 hr of LPS stimulation (FIGS. 9A and 9B).

To investigate the pituitary expression of MIF in vivo, pituitary mRNAanalysis was performed in mice injected with sublethal amounts of LPS(2.25 mg/kg). Pituitary RNA was isolated at increasing time intervalsand subjected to reverse transcription-polymerase chain reaction(RT-PCR) analysis. Pituitary MIF mRNA levels increased with time andreached a plateau 16-24 h after LPS challenge in endotoxin-sensitivemice (FIG. 10). These findings were confirmed by competitive PCRanalysis of representative cDNA preparations that showed a 3-foldincrease in pituitary MIF mRNA after LPS stimulation (FIG. 11). Althoughinfiltrating mononuclear cells were not evident in stained pituitarysections obtained after LPS treatment, in order to exclude a potential Tcell contribution of pituitary MIF mRNA, pituitary cDNA was amplifiedwith primers specific for the T cell-specific gene product, CD2. Despiteintentional overcycling, RT-PCR analysis of CD2 was uniformly negative,ruling out infiltrating T cells as a possible source of pituitary MIFmRNA (FIG. 10). LPS did not induce pituitary MIF mRNA in the geneticallyendotoxin-resistant strain C3H/HeJ, while congenic endotoxin-sensitiveC3H/HeN mice showed an increase of pituitary MIF mRNA between 0 hr and24 hr similar to that observed with BALB/c mice.

The pituitary content of MIF protein in vivo was analyzed by Westernblotting of pituitary lysates. Pituitaries obtained from normal,non-stimulated mice showed significant amounts of pre-formed MIF protein(FIG. 12). A significant decrease in pituitary MIF content was observed8-20 h after LPS injection in endotoxin-sensitive mice, but not inendotoxinresistant (C3H/HeJ) mice.

Previous studies over the years have identified MIF to be a product oflectin-stimulated T cells (Weiser et al., 1989, Proc. Natl. Acad. Sci.USA 86:7522-7526; Weiser et al., 1981, J. Immunol. 126:1958-1962). SinceLPS does not directly activate T cells, MIF that appears in thecirculation presumably results from direct stimulation of various celltypes, including but not limited to pituitary cells andmonocyte/macrophages by LPS or from indirect activation LPS-inducedmediators of these and other cell types, including for instance T cells.In order to better establish the contribution of various cell types tocirculating serum MIF in endotoxemic mice, control BALB/c, and Tcell-deficient BALB/c^(nu/nu) mice were injected intraperitoneally withLPS, serum samples were analyzed by gel electrophoresis and Westernblotting, and serum MIF levels quantified by laser densitometry (FIG.13). Serum MIF was detected at 2 h in wild-type mice (BALB/c) andincreased over 20 h in a time-dependent fashion. In contrast, Tcell-deficient (nude) mice showed a markedly delayed response in theappearance of serum MIF. MIF levels increased significantly only after 8h of LPS challenge.

These findings indicate that pituitary MIF contributes directly tocirculating serum MIF that is detectable in the post-acute phase (≧8hours) of endotoxaemia. This time course is consistent with thedisappearance of pituitary MIF protein and the induction of pituitaryMIF mRNA levels that were observed in vivo (FIGS. 10 and 11). Theresults obtained in nude mice indicate that T cell MIF contributesprimarily to the serum MIF that appears in the first 8 h. LPS treatment(2.25 mg/kg) of endotoxin-resistant C3H/HeJ mice produced no detectableserum MIF.

12. EXAMPLE Production of Migration Inhibitory Factor by Macro Phages

12.1. Materials and Methods

12.1.1. Reagents

E. coli 0111:B4 LPS, polymyxin B, carbenicillin, PMSF and Tween-20 wereobtained from Sigma (St. Louis, Mo.). LPS was resuspended inpyrogen-free water, vortexed, sonicated, aliquoted (5 mg/ml), and storedat −20° C. Serial dilutions of LPS were prepared in pyrogen-free waterby sonication (Branson 3210, Danbury, Conn.). Gentamicin was from Gibco(Grand Island, N.Y.). Thioglycollate broth (Difco, Detroit, Mich.) wasprepared according to the manufacturer's recommendation, autoclaved, andstored protected from light at room temperature. Horseradishperoxidase-conjugated goat anti-rabbit antibody was purchased fromPierce (Rockford, Ill.) and 4-chloro-1-naphthol and stabilized3,3′,5,5′-tetramethylbenzidene (TMB) substrate for horseradishperoxidase were from Promega (Madison, Wis.). Polyclonal anti-MIF serumwas generated by immunizing New Zealand White rabbits (Hare Marland,Hewitt, N.J.) with purified recombinant murine MIF. On week 1 and 2,rabbits were inoculated intra-dermally with 100 μg of rMIF diluted incomplete Freund's adjuvant, and with 50 μg of rMIF diluted in incompleteFreund's adjuvant on week 4. Immune serum was collected one week afterthe last inoculation.

12.1.2. Cytokines

Recombinant murine MIF (rMIF) was expressed in E. coli BL21/DE3(Novagen, Madison, Wis.) and purified to homogeneity by anion exchange(Mono Q; Pharmacia, Piscataway, N.J.) and reverse phase chromatography(C8-SepPak, Millipore, Milford, Mass.), lyophilized and reconstituted insodium phosphate buffer (20 nM, pH 7.2) following procedures in Example6, supra. MIF bioactivity was established by measuring dose-dependentMIF-induced augmentation of Leishmania major killing by macrophages andby neutralization of this activity with anti-MIF antibody. rMIFcontained 9 pg of endotoxin per μg protein as determined by thechromogenic Limulus amoebocyte assay (Bio-Whittaker Inc., Walkersville,Md.). Recombinant murine IL-113 and IL-6 (5 μg/ml after reconstitution)were obtained from R&D (Minneapolis, Minn.) and recombinant murine IFNγ(10⁵ IU/ml) was from Boehringer-Mannheim (Indianapolis, Ind.). Cytokineswere reconstituted in pyrogen-free water containing 0.1% of very lowendotoxin BSA (Miles Inc., Kankakee, Ill.) and stored at −80° C. Theendotoxin content of the reconstituted cytokines was 0.5 ng/μg of TNFα,2 ng/μg of IL-1β, 1.1 ng/μg of IL-6 and 0.06 pg/unit of IFNγ asdetermined by the chromogenic Limulus amoebocyte assay.

12.1.3. Animals and Experimental Design

Eight to 10-week-old (19-21 g) female BALB/c (control), T cell-deficientBALB/c^(nu/nu) (nude), and hypophysectomized BALB/c mice were purchasedfrom Charles River (Kingston, N.Y.). Animals were housed in groups of 5or 10 mice per cage with free access to food and water (supplementedwith 5% glucose for hypophysectomized mice) and were acclimatized for 5days prior to experimentation. Mice were injected i.p. with nonlethaldoses of LPS (2.25 mg/kg for BALB/c and T cell-deficient mice, and 50μg/kg for hypophysectomized mice), and sacrificed 2, 8 and 20 h afterLPS challenge to collect serum. The LPS dose was adjusted to provide acomparable degree of lethality in the control BALB/c group as among thehypophysectomized mice, which are hypersensitive to endotoxin. Five μlof serum was analyzed by Western blotting and visualized with anti-MIFantibody.

12.1.4. Cells

RAW 264.7 murine macrophages, THP-1 human monocytes, ASL-1 murine andJurkat human T lymphocyte cell lines were obtained from the AmericanType Culture Collection (Rockville, Md.). Cells were grown in RPMI 1640medium (Gibco, Grand Island, N.Y.) containing 2 mM glutamine, 10%heat-inactivated fetal bovine serum (FBS) (HyClone, Logan, Utah), 50μg/ml of carbenicillin and gentamicin. Medium for ASL-1 cellsadditionally was supplemented with 1 mM sodium pyruvate.Thioglycollate-elicited peritoneal macrophages were obtained from BALB/cmice that were injected i.p. 3-4 days previously with 2 ml of sterilethioglycollate broth. Cells were harvested under strict asepticconditions by lavage of the peritoneal cavity with 5 ml of anice-chilled 11.6% sucrose solution. After centrifugation (10 min at 800g), cells were resuspended in RPMI/10% FBS, enumerated, and plated at adensity of 2×10⁶ or 4×10⁶ cells/well. Human polymorphonuclear leukocyteswere isolated from peripheral blood by density gradient centrifugation(Vadas et al. 1979, J. Immunol. 122: 1228; Sherry et al., 1981, In:Methods for Studying Mononuclear Phagocytes, Academic Press, NY, p.187).

12.1.5. MIF Content of Macrophage and T Lymphocyte Lysates

Aliquots of 1×10⁶ cells of each type were lysed with Tris-bufferedsaline(50 mM Tris-Base, 150 mM NaCl, pH 7.5) containing 1% NP-40, 0.5%deoxycholic acid, 0.1% SDS and 2 mM EDTA. Cellular debris was pelletedand the supernatants were diluted with an equal volume of reducingSDS-PAGE sample buffer. Ten microliter of lysate (equivalent to 5×10³cells) were electrophoresed through 18% polyacrylamide gels, andtransferred to nitrocellulose membranes for Western blot analysis usingpolyclonal rabbit anti-MIF serum.

12.1.6. Stimulation of Macrophages by LPS and Cytokines

RAW 264.7 macrophages were washed with fresh medium, harvested by gentlescraping, resuspended in RPMI/10% FBS, and incubated at 2×10⁶ or 4×10⁶cells/well in 3.5-cm tissue culture plates (Linbro®, Flow, McLean, Va.).After 3 h of incubation at 37° C. in a humidified atmosphere with 5%CO₂, nonadherent cells were removed and wells were washed twice withRPMI/1% FBS. Cells then were incubated for 12 h with LPS (atconcentrations ranging from 1 pg/ml to 1 μg/ml) or with cytokines (1 or10 ng/ml for TNFα, IL-1β, and IL6, and 10, 100 or 1000 IU/ml for IFNγ)diluted in RPMI/1% FBS. For time-course experiments, conditioned mediaof parallel cultures were removed at 3, 6, 9, 12 and 15 h intervalsafter LPS stimulation. Thioglycollate-elicited peritoneal macrophages(10⁷ cells/well) were harvested as described and conditioned similarlyto RAW 264.7 cells. Cells were incubated for 12 h either with LPS orwith IFNγ plus LPS. When co-stimulated with IFNγ and LPS, cells werefirst incubated for an hour with IFNγ (100 IU/ml) before addition of LPSat the indicated concentrations. At the end of each experiment,cell-culture media were collected, centrifuged (10 min at 800 g), andsupplemented with PMSF (1 mM). Supernatants then were concentrated10-fold by membrane filtration (10 kD cut-off) (Centricon-10, Amicon,Beverly, N.Y.). Cellular RNA was extracted from adherent cells.

12.1.7. Reverse Transcription and Polymerase Chain Reaction CRT-PCR)

Total cellular RNA was extracted from macrophages with RNAzol-B(Tel-Test Inc., Friendswood, Tex.) following the manufacturer'sprotocol. One μg of RNA was reverse transcribed using oligo (dT)₁₂₋₁₈and M-MLV reverse transcriptase (Gibco, Grand Island, N.Y.) in a 50 μlreaction. Five μl of cDNA was amplified by PCR in a Perkin-Elmer/Cetus9600 thermal cycler (denaturation 1 mM at 94° C., annealing 1 mM at 55°C., elongation 1 min at 72° C.) using murine MIF primers (32 cycles),TNFα primers (22 cycles), or β-actin primers (25 cycles).Intron-spanning TNFα primers were 5′-GCGGAGTCCGGGCAGGTCTA-3′ [SEQ IDNO:16] and 3′-GGGGGGCTGGCTCTGTGAGG-5′ [SEQ ID NO:17]. DNA amplificationproducts were analyzed on 2% agarose gels and gel loading was normalizedto β-actin PCR products. Quantification of MIF mRNA from LPSstimulatedRAW 264.7 macrophages was done by competitive PCR (Gilliland et al.,1990, Proc. Natl. Acad. Sci. USA 87: 2275). The 273 bp competitive cDNAtemplate was added at the indicated concentrations and co-amplified withMIF cDNA using MIF primers.

12.1.8. Western Blots

For Western blotting, samples were resolved on 18% SDS polyacrylamidegels under reducing conditions and transferred onto nitrocellulosemembrane (Schleicher and Schuell, Keene, N.H.) at 50 V and 150 mA for 16h. Membranes were blocked with 500 mM NaCl, 50 roM Tris-Base, pH 7.5buffer containing 5% nonfat dry milk and 0.05% Tween-20 and incubatedfirst with polyclonal rabbit anti-rMIF serum and then with horseradishperoxidase-conjugated goat anti-rabbit IgG antibody (each diluted 1:1000in 500 mM NaCl, 50 mM Tris-Base, pH 7.5 buffer with 1% BSA and 0.05%Tween-20). Polyclonal rabbit anti-murine MIF serum was demonstrated toreact with both natural and recombinant murine and human MIF.Incubations were for 1 h each. MIF was visualized by development withchloronaphthol/H₂O₂ or with TMB substrate. Serum MIF bands werequantified by laser densitometry (Shimadzu FDU-3, CS-9000U, Braintree,Mass.). Faint densitometric signals in the MIF region were detected inmost sera prior to LPS administration (0 h). Hence, scanning integralsare presented as relative serum MIF content (i.e. fold-increase)normalized to the respective 0 h zone (i.e. background or baselinestaining) on each membrane. Each plotted point is the mean±SEM ofindividual sera from 2 to 5 animals.

12.1.9. MIF-Induced Secretion of TNFα by Macrophages

Cells (4×10⁶/well) were processed as described above and stimulated for12 h with rMIF at specified concentrations ranging from 100 pg/ml to 1μg/ml. At 1 μg/ml of rMIF, LPS contamination was 9 pg/ml as assessed byLimulus assay. To neutralize this minute amount of LPS, rMIF waspreincubated for 1 h at 37° C. with polymyxin B (1 μg/ml). MIF-inducedTNFα activity in cell-culture supernatant was quantitated by L929 cellcytotoxicity (Wolpe et al., 1988, J. Exp. Med. 167: 570). MIF-inducedTNFα activity was blocked completely by anti-TNFα antibodies. rMIF didnot contribute to TNFα activity, as recombinant murine TNFα (dose range:5 pg/ml to 1 μg/ml) cytotoxicity remained unchanged when rMIF (10 pg/mlto 10 μg/ml) or anti-MIF polyclonal serum were added to L929 cellstogether with rTNFα.

12.2. Results

12.2.1. Serum MIF Levels in LPS-Injected Mice

Pituitary MIF contributes significantly to the MIF that appears in serumduring endotoxemia. Serum MIF reached peak levels in normal mice at 20 hbut was undetectable in hypophysectomized mice at this time,demonstrating that the pituitary is a major source of the MIF thatappears in serum after LPS injection.

The central role played by MIF in endotoxic shock, together with theknown hypersensitivity of hypophysectomized mice to LPS, led toexperiments to examine more closely the kinetics of MIF appearance inthe circulation during the early, acute phase (i.e. 2-8 h) ofendotoxemic shock. For comparison purposes, serum MIF kinetics were alsoexamined in nude mice, which lack a T cell source of MIF. Control,hypophysectomized, and nude mice were injected with LPS and serumprepared at intervals and quantified for MIF content by Western blottingand laser scanning densitometry (FIG. 14). Whereas serum MIFconcentrations increased gradually over 20 h in control mice,hypophysectomized mice exhibited a rapid rise and then prompt fall inMIF that peaked 2 h after LPS injection. In nude mice, the rise in serumMIF was delayed by 8 to 12 h, but increased in a manner that was similarto that of the control mice. Given the lack of a pituitary source of MIFin the hypophysectomized mice, the MIF present in the circulation at 2 hreflected the release of MIF by an additional LPS-sensitive cellpopulation. Such serum MIF kinetics were reminiscent of a macrophageTNFα response (Beutler et al., 1985, J. Exp. Med. 161: 984; Beutler etal., 1985, J. Immunol. 135: 3972; Michie et al., 1988, N. Eng. J. Med.318: 1481) and thus the possibility that MIF was produced by cells ofthe monocyte/macrophage lineage was examined.

12.2.2. Expression of MIF in Resting Macrophages

Since both a pituitary cell-line (AtT-20) and the whole pituitary invivo contain substantial amounts of pre-formed MIF, the intracellularMIF content of various inflammatory cells and cell lines was analyzed.Significant amounts of pre-formed MIF protein were found to be presentin cell lysates obtained from resting, non-stimulated murine RAW 264.7cells, murine peritoneal macrophages, and human THP-1 monocytes (FIG.15). It was estimated that on average there was 0.1-1 pg ofimmunoreactive, pre-formed MIF protein per macrophage. The MIF contentof murine or human monocytesjmacrophages was similar to that of two Tcell lines, the human Jurkat and the murine ASL-1. In contrast, celllysates obtained from purified polymorphonuclear leukocytes (PMNs)exhibited no detectable MIF protein (FIG. 15).

12.2.3. MIF Secretion by LPS-Stimulated Macrophages

MIF was observed to be secreted by macrophages into culture medium afterLPS stimulation. In RAW 264.7 macrophages, MIF secretion was induced byas little as 10 pg/ml of LPS, peaked at 1 ng/ml, and was not detectableat LPS concentrations>1 μg/ml (FIG. 16A). In elicited peritonealmacrophages, 1 ng/ml of LPS was required to induce MIF secretion, and amaximal response was observed with 10-100 ng/ml of LPS (FIG. 16B). Ofsignificance, co-stimulation of cells with IFNγ (100 IU/ml, given 1 hprior to LPS) plus LPS resulted in a >1000-fold increase in LPSresponsiveness (FIG. 16C).

We next examined the time-course of MIF secretion in parallel culturesof RAW 264.7 macrophages maximally stimulated with 1 ng/ml of LPS. Asassessed by Western blotting, MIF was first detected 6 to 9 h after LPSstimulation. Maximum amounts of MIF appeared in medium between 9 and 12h post-LPS stimulation. MIF levels then decreased, indicating that by 12h, MIF is removed or degraded at a rate that exceeds that of synthesisand release by macrophages.

12.2.4. Expression of MIF mRNA by LPS-stimulated macro phages

The expression of MIF mRNA by LPS-stimulated RAW 264.7 macrophages wasinvestigated by reverse transcription and polymerase chain reaction(RT-PCR). Parallel cultures were incubated for 12 h with medium orincreasing amounts of LPS and analyzed for the expression of MIF, TNFα,and β-actin. MIF mRNA was expressed constitutively in non-stimulatedmurine RAW 264.7 cells (FIG. 17) and in elicited peritoneal macrophages.As expected, the expression of TNFα mRNA increased over the range of LPSconcentrations (1 pg/ml to 1 μg/ml). In contrast, MIF mRNA levelscorrelated inversely with LPS concentration over this dose range. MIFmRNA level was highest in cells induced with 1 pg/ml of LPS and lowestin those induced by 1 μg/ml of LPS.

To assess more quantitatively the induction of MIF mRNA, cDNA wasprepared from control and LPS-stimulated cultures of RAW 264.7macrophages and analyzed by competitive PCR. The amount of competitivetemplate which was required to obtain equivalent levels of MIF andcompetitor DNA amplification products was 1.5 pg for non-stimulated(control) macrophages and 3 pg for LPS-induced (100 pg/ml) RAW 264.7macrophages (FIG. 18). Therefore, macrophage MIF mRNA increasedapproximately 2-fold after LPS induction, an increase comparable to thatobserved in pituitary cells in vivo (3-fold after induction with 45 μgof LPS injected i.p.). The time-course of MIF mRNA induction wasexamined in RAW 264.7 macrophages incubated with 1 ng/ml of LPS, the LPSconcentration which induced maximum MIF secretion in these cells. MIFmRNA levels increased 6 h after LPS induction and remained elevated forup to 12 h post-LPS.

12.2.5. Interaction Between MIF and Pro-Inflammatory Cytokines

The detection of elevated levels of MIF in blood after LPSadministration together with the finding of an important role for MIF inexperimental endotoxemia led to the investigation of the interactionbetween MIF and other pro-inflammatory cytokines. MIF release by RAW264.7 macrophages was studied after stimulation with recombinant murineTNFα, IL-1β, IL-6, or IFNγ. By Western blotting, TNFα and IFNγ werefound to induce MIF secretion in a dose-dependent fashion (FIG. 19). Thelowest concentration of cytokine which was effective under theseexperimental conditions was 1 ng/ml of TNFα and 10 IU/ml of IFNγ. TheTNFα and IFNγ effects could not be accounted for by LPS contamination ofrecombinant cytokine preparations, which were found to be 0.5 pg/ng ofTNFα and 60 fg/U of IFNγ. LPS and IFNγ had additive/synergistic effecton MIF secretion, whereas TNFα and IL-1β did not. Finally, MIFproduction was not induced by either IL-16 or IL-6 at 1 or 10 ng/ml.

The secretion of TNFα and IL-1β by RAW 264.7 macrophages was examinedafter stimulation with rMIF. MIF samples were pre-incubated for 1 h withpolymyxin B at 1 μg/ml to neutralize small amounts of contaminating LPS(9 pg/μg of MIF by the chromogenic Limulus assay). rMIF atconcentrations ≧100 ng/ml was found to induce the secretion of bioactiveTNFα as determined by the L929 cytotoxicity assay (FIG. 20). TheMIF-induced TNFα bioactivity was blocked completely by anti-TNFαantibodies and rMIF did not contribute to TNFα cytotoxicity, asrecombinant murine TNFα cytotoxicity was unchanged when rMIF or anti-MIFpolyclonal serum were added to L929 cells together with rTNFα. IL-1βsecretion, in contrast, was not detectable by Western blotting over theconcentration of rMIF tested.

13. EXAMPLE Steroid Induces MIF Secretion by Macrophages

13.1. Materials and Methods

13.1.1. Cell Preparation and MIF Induction

RAW 264.7 murine macrophages were obtained from the American TypeCulture Collection (Rockville, Md.). Cells were grown in RPMI 1640medium (Gibco, Grand Island, N.Y.), 10% heat-inactivated fetal bovineserum (FBS) (HyClone, Logan, Utah), 50 μg/ml of carbenicillin andgentamicin. RAW 264.7 macrophages were washed with fresh medium,harvested by gentle scraping, resuspended in RPMI/10% FBS, and incubatedat 3×10⁶ cells/well in 3.5-cm tissue culture plates (Linbro™, Flow,McLean, Va.). After 3 h of incubation at 37° C. in a humidifiedatmosphere with 5% CO₂, nonadherent cells were removed and walls werewashed twice with RPMI/1% FBS. In some experiments designed to test forinhibition of MIF release, test compounds were then added to the culturemedia. For example, the cells were pre-treated with 20α-dihydrocorticolat a final concentration of 10⁻⁶M. Cells then were incubated for 12-15 hwith various steroids at concentrations ranging from 10⁻⁶ m to 10⁻¹⁴M(e.g., dexamethasone), along with the desired test compound (e.g.,20α-dihydrocortisol) if any. At the end of experiment, cell culturemedia were collected and centrifuged (10 min at 800 g). Cell-conditionedmedia were concentrated 10-fold by membrane filtration (10 kDa cut-off)(Centricon-10, Amicon, Beverly, N.Y.).

Thioglycollate-elicited peritoneal macrophages were obtained from BALB/cmice that were injected i.p. 3-4 days previously with 2 ml of sterilethioglycollate broth. Cells were harvested under strict asepticconditions by lavage of the peritoneal cavity with 5 ml of anice-chilled 11.6% sucrose solution. After centrifugation (10 min at 800g), cells were resuspended in RPMI/10% FBS, enumerated and plated at adensity of 3−4×10⁶ cells/well. Cells were incubated for 12 to 15 h withvarious steroids (at concentrations ranging from 10⁻⁶M to 10⁻¹⁴M).

Samples were resolved on 18% SDS polyacrylamide gels under reducingconditions and transferred onto nitrocellulose membrane (Schleicher andSchuell, Keene, N.H.) at 50 V and 150 mA for 16 h. Membranes wereblocked with 500 mM NaCl, 50 mM Tris-Base, pH 7.5 buffer containing 5%nonfat dry milk and 0.05% Tween-20 and incubated first with polyclonalanti-rMIF serum and then with horseradish peroxidase-conjugated goatanti-rabbit IgG antibody (each diluted 1:1000 in 500 mM NaCl, 50 mMTris-Base, pH 7.5 buffer with 1% BSA). Polyclonal rabbit anti-murine MIFwas shown to react with both natural and recombinant murine and humanMIF. Incubations were 1 h each. MIF was visualized by development withchloronaphtol/H₂0₂ or with TMB substrate.

13.1.2. Steroid Injection

Canulated Sprague-Dawley female rats were purchased from Zivic-Miller.Animals were housed with free access to food and water and wereacclimatized for at least 2 days prior to experimentation. Rats wereinjected i.v. through the jugular vein with either dexamethasone (1, 10or 20 mg/kg), or with RU38486 (mifepristone) (1 or 10 mg/kg), or withthe two drugs together (both at 10 mg/kg, RU38486 was injected 15 min.before dexamethasone). Serum was collected 3, 6, and 9 hourspost-steroids injection and analyzed by Western blotting with anti-MIFantibody.

13.2. Results

MIF was observed to be secreted by murine macrophages (RAW 264.7macrophages and/or elicited peritoneal macrophages) after stimulationwith dexamethasone, hydrocortisol,5β-Pregnane-3α,11β,17α,21-tetrol-20-one (tetrahydrocortisol),4-pregnene-11β,17α,20α,21-tetrol-3-one (20α dihydrocortisol), RU38486(mifepristone), aldosterone, testosterone, and progesterone (Table V).In addition, dexamethasone at 10 mg/kg, RU 38486 at 10 mg/kg or acombination of both drugs induced MIF secretion in vivo. Serum MIF wasdetected 3 to 6 hours after steroid injection.

As shown in FIG. 21, pretreatment and co-administration of20α-dihydrocortisol attenuates or abolishes the MIF response todexamethasone.

TABLE V STEROIDS INDUCE MIF SECRETION BY MURINE MACROPHAGES Range ofSteroid Concentrations (M) Inducing MIF in: RAW 264.7 PeritonealSteriods Macrophages macrophages Dexamethasone 10⁻⁸ to 10⁻¹⁴ 10⁻⁸ to10⁻¹² Hydrocortisone 10⁻⁸ to 10⁻¹² 10⁻⁸ to 10⁻¹⁶ 5β-Pregnane- 10⁻⁸ to10⁻¹⁴ Not tested 3α,11β,17α,21- Tetrol-20-one (Tetrahydrocortisol)4-Pregnane- 10⁻¹⁰ to 10⁻¹⁴  10⁻¹⁰ to 10⁻¹⁴  11β,17α,20α,21-tetrol-3-one(20α dihydrocortisol)* Aldosterone 10⁻¹⁰ to 10⁻¹⁴  10⁻⁸ to 10⁻¹⁴Testosterone 10⁻⁶ to 10⁻¹⁴ 10⁻⁸ to 10⁻¹⁴ RU-38486 (mifepristone)* 10⁻⁸to 10⁻¹⁴ Not tested Progesterone 10⁻⁴ to 10⁻¹² Not tested *At higherconcentrations, these steroids inhibit the macrophage release of MIF inresponse to a challenge does of dexamethasone, i.e., a dose thatnormally would induce MIF release.

14, EXAMPLE Organ Distribution of MIF and MIF Receptor

The following example describes the organ distribution of MIF and MIFreceptor in mice.

14.1. Materials and Methods

14.1.1. Labeling of Exogenous MIF

Five μl ¹²⁵I-MIF (specific activity, 2×10⁴ cpm/ng) together with 5μl⁵¹Cr-labeled red blood cells (RBCs), were injected intravenously in atotal of 300 μl saline. One preparation additionally contained 40 μgunlabeled MIF as a competitor. Ten minutes prior to injection, eachmouse was given 10 mg Na-iodide i.p. Mice were sacrificed 10 minutesafter injection.

14.1.2. Tissue Preparation

Four hundred μl blood was collected from each mouse immediately aftersacrificing. Additionally, various organs were removed, rinsed in PBS,and blotted dry on filter paper. Organs were weighed and radioactivitywas counted on two separate windows (one for ¹²⁵I, one for ⁵¹ Cr) on agamma counter. The remainder of the carcass was also weighed andcounted.

14.1.3. Organ Distribution of Endogenous MIF

The organ distribution of MIF protein was examined in a 8 to 10-week oldfemale BALB/c mouse sacrificed by CO₂ asphyxiation and necropsied underaseptic conditions. Organs (brain, liver, spleen, kidneys, adrenals andlungs) were excised, sectioned and homogenized at 4° C. withTris-buffered saline (50 mM Tris-Base, 150 mM NaCl, pH 7.5) containing1% NP-40, 0.5% deoxycholic acid, 0.1% SDS, 2 mM EDTA and 1 mH PMSF.Cellular debris was pelleted and aliquots of organ lysate supernatants,adjusted for protein concentration, were diluted with an equal volume ofreducing SDS-PAGE sample buffer. Samples (equivalent to 60 μg ofprotein) were electrophoresed through 18% polyacrylamide gels andtransferred to nitrocellulose membranes for Western blot analysis usingpolyclonal anti-MIF serum.

14.2. Results

The organ distribution of endogenous MIF protein was examined in normalmice. Mouse tissues were homogenized and aliquots of total proteinelectrophoresed and analyzed by Western blotting. As shown in FIG. 22,pre-formed MIF was detectable in mouse liver, spleen, kidney and brain.Much lower amounts were also found in the adrenals and lungs. Theseorgans contain significant amounts of macrophages, suggesting that theMIF protein present in these tissues is at least partlymacrophage-associated.

¹²⁵I-MIF was also injected into mice in order to determine the tissuedistribution of MIF receptors. The index of the tissue specificlocalization of radiolabelled MIF was determined, as described inBeutler et al., 1985, J. Immunol. 135:3972. As shown in Table VI, aparticularly high level of MIF-binding was observed in the kidneys andliver among the organs examined.

TABLE VI Index of Tissue Specific Localization of Organs RadiolabelledMIF Liver 256 Kidney 2334 Lung 52 Adrenal 47 Spleen 78 Small Intestine247 Large Intestine 122 Brain 17 Skin 103

15. EXAMPLE MIF Receptor Identification

In this example, receptor proteins that bind to MIF are identified, andthe partial amino acid sequences of each of these MIF receptors arepresented.

15.1. Materials and Methods

15.1.1. Cell Lysate Preparation

Two large tissue culture plates (5×10⁷ cells) of murine RAW 264.7 cells(a monocyte-like line) were solubilized by the addition of 2.5 ml oflysis buffer (50 mM Tris pH 7.5, 150 mM NaCl, 1% Triton X-100, withprotease inhibitors). Plates were incubated on ice with gentle rotation.Plates were then scraped, the contents collected, and centrifuged in 4microfuge (1.5 ml) tubes at 10,000×g for 10 min at 4° C. Supernatantswere collected and stored on ice.

15.1.2. Affinity Chromatography

For preparation of an MIF-conjugated affinity column, 1 gm ofCNBr-Sepharose was first washed with 1 mM HCl. Bacterial recombinantmurine MIF (3 mg) was dissolved in 40% acetonitrile/H₂0. MIF solutionthen was mixed with activated CNBr-Sepharose and incubated for 3 hr atroom temperature. Unreacted CNBr-Sepharose then was quenched byincubation with 0.2 M glycine pH 8.0 overnight. Coupling efficiency wasdetermined by comparison of optical absorbance of MIF solution beforeand after coupling, and determined to be 85%. Resin then was washed,poured into a column and equilibrated with lysis buffer.

Supernatants of lysed RAW cell preparation were loaded ontoMIF-Sepharose at a flow rate of 2 ml/hr, and washed sequentially with 3column volumes of lysis buffer. Remaining bound protein was eluted with5 volumes of 0.05% Triton X-100 in 0.1 M glycine, pH 2.5. Fractions werecollected and buffered with 90 μl of 1 M Tris base.

15.1.3. Gel Electrophoresis

Fractions containing protein species of interest were concentrated overCentricon-10 membranes, resuspended in equal volumes of 2× Laemmlisample buffer, and electrophoresed on 7.5% SDS-PAGE gels. Proteins weretransferred onto PVDF filters utilizing 10 mM CAPS pH 11/20% methanoltransfer buffer. Filters were stained with Ponceau-S and protein bandscorresponding to molecular weights 52 kD and 72 kD were identified andexcised.

15.1.4. Microsequencing Analysis

The excised protein bands were subjected to microsequencing analysisusing automated gas-phase sequencing methods (Applied Biosystems).

15.2. Results

Putative MIF receptor protein was isolated by MIF affinitychromatography of RAW cell lysates on an MIF affinity column, asdescribed above in Section 13.1.2.

Three fractions (6, 7, 8) were found to contain two protein species, 52kD and 72 kD, when analyzed by SDS-PAGE. When parallel transfers wereperformed onto membranes which were then incubated with ¹²⁵I-labelledMIF, it was demonstrated that both the 52 kD and the 72 kD bands boundradiolabelled MIF. Ligand binding was inhibited by simultaneousincubation of membranes with excess unlabelled MIF, thereby showing thatbinding was MIF specific.

Protein from the 52 kD species was isolated and subjected to amino acidmicrosequencing. A partial amino acid sequence was obtained from this 52kD putative MIF receptor, as shown here:

[SEQ ID NO: 7] Ile-X-His-Asn-Thr-Val-Ala-Thr-Glu-Ile-(Ser)-(Gly)Tyr-Asn-(Asn/Gly)-(Ala)-(Met)The residues in parenthesis are tentative designation.

Similarly, protein from the 72 kD species was isolated and sequenced. Apartial amino acid sequence was determined and shown here:

[SEQ ID NO: 6] Ala-Lys-Lys-Gly-Ala-Val-Gly-Gly-Ile

16. EXAMPLE Inhibition of Humoral Immunity with Anti-MIF Antibody

Treatment of naive animals with purified neutralizing anti-MIF antibodywas shown to inhibit the development of a primary immune response to asoluble test antigen, e.g. ribonuclease (RNase). Five BALB/c mice wereimmunized by intraperitoneal injection with 0.1 mg of bovine RNase A(dissolved in 0.1 ml sterile PBS) emulsified with complete Freund'sadjuvant (1:1). This was followed by the intraabdominal injection of 0.4mg polyclonal anti-MIF IgG (in 0.1 ml PBS) or, in control mice, 0.4 mgof purified pre-immune IgG. The antibodies were administered again at 3,5 and 7 days after the primary immunization. After 12 days, the animalswere sacrificed and blood samples were collected for serum assays foranti-RNase immunoglobulin by ELISA with RNase-coated plates. Whencompared to RNase-immunized controls, anti-MIF treated RNase-immunizedmice showed a 70% reduction in circulating anti-RNase immunoglobulinlevels (n=5 mice per group).

17. EXAMPLE Production of Monoclonal Antibodies Directed Against Humanand Murine MIF

Hybridomas secreting monoclonal antibodies (MAbs) directed against humanand murine forms of MIF were made and isolated according to methods wellknown in the art. In brief summary, female BALB/c mice were immunizedintraperitoneally (i.p.) with recombinant murine or human MIF (10μg/mouse) in Ribi Adjuvant (Ribi Immunochem.). During the immunizationand boost period, mice were tail-bled and serum anti-MIF antibodytiters, as well as isotype distribution (IgM vs IgG), were assayed bymicrotiter plate-based direct enzyme-linked immunosorbent assay (ELISA)methods on wells with immobilized recombinant MIF (250 ng/ml; 55μl/well) as antigen. Immunized mice were given booster injections ofrecombinant MIF (10 μg/mouse) in Ribi Adjuvant at least four timesbefore spleens were removed for fusion. Three days before spleen cellfusion with mouse myeloma cells (P3X63Ag8.653; American Type CultureCollection) using polyethylene glycol (Boerhinger Mannheim), mice wereboosted i.p. with both murine and human MIF (10 μg in PBS). Hybridomaswere expanded under HAT (hypoxanthine, aminopterin, and thymidine;GIBCO) selection medium (DMEM containing HAT, 10% Condimed (BoerhingerMannheim), 20% FBS (Hyclone), and antibiotics (penicillin, streptomycin;GIBCO) for two to three weeks. Culture supernatants from growinghybridomas were screened for anti-MIF antibodies by direct ELISA methodswith immobilized recombinant MIF.

Immunoreactivity of antibodies from anti-MIF positive clones was furtheranalyzed by Western immunoblotting techniques, and high-titer producinghybridomas were chosen for re-cloning by limiting dilution. Anti-MIFmonoclonals were isotyped using Screentype ELISA (Boehringer Mannheim).Hybridomas secreting desired monoclonal antibodies (IgG-type) were grownas ascites in BALB/c mice, and MAb's were purified using T-gelchromatography (Pierce). Several IgM-type anti-MIF monoclonal antibodieswere identified but not further characterized. Several IgG-secretinghybridomas were isolated and characterized (Table VII).

TABLE VII Reactivity with Human MurineIgG MIF MIF Subtype VIIG3 − +IgG2b IXD11 − + IgG2a XB2 − + IgG3 XID5 − + IgG2b XIG2 − + IgG3 VD8 − +IgG2b IID9 + + IgG1 IIID9 + + IgG1 XIF7 + + IgG2b I31 + + IgG1 IV2.2 + +IgG1 XI7 + + n.d. XII15.6 + + IgG1 XIV15.4 + + IgG1

17.1 Test for Anti-MIF Neutralization Activity

Purified anti-MIF monoclonal antibodies were first tested forneutralization activity in a macrophage killing assay.Thioglycollate-elicited mouse peritoneal macrophages were obtained fromBALB/c mice, allowed to adhere for 4 hours, and then infected with theintracellular parasite Leishmania major at a parasite:macrophage ratioof 8:1. After washing, infected macrophage cultures were treated withrecombinant human MIF (which enhances macrophage-killing ofintracellular parasites in a dose-dependent fashion when compared toculture medium controls) with or without added VIIG3 or XID5 monoclonalanti-MIF antibodies (25 μg/ml). Both antibodies were found to neutralizethe MIF-enhanced killing of L. major by about 50%.

In separate experiments, purified monoclonal anti-MIF antibodies weretested for MIF neutralizing activity in a [³H]-thymidine incorporationassay with primary murine T cells cultured on anti-CD3 IgG-coated(Pharmingen) tissue culture plates. Briefly described, this assayemployed BALB/c spleen cells that were isolated using murine T cellenrichment columns (R&D) and grown on anti-CD3 IgG-coated 96 wellmicro-titer plates in RPMI containing 10% FBS, antibiotics (penicillin,streptomycin) and L-glutamine together with anti-MIF or control mousemonoclonals antibodies. After 48 hours, T cells were pulsed with[³H]-thymidine for 16 to 18 hours, harvested and counted bybeta-scintillation counting methods. As a positive control, anti-IL-2monoclonal antibodies (Genzyme) were added to inhibit proliferation andassociated [³H]-thymidine incorporation. Both the VIIG3 and the XID5antibodies decreased thymidine incorporation by about 20%; anti-IL-2treatment reduced [³H]-thymidine incorporation by about 75%.

17.2. Development of Quantitative Sandwich ELISA for MIF

A MIF-specific “sandwich” ELISA technique was developed, based on thetrapping of MIF by immobilized VIIG3 antibody followed by detection witha rabbit polyclonal anti-MIF antiserum. This assay was performed asfollows:

Immulon II (Dynatech) ELISA plate wells were coated with 10-15 μg/ml MAb(VIIG3) in PBS (65 μl/well); the MAb had been purified from ascitesusing T-gel absorbant (Pierce). Plates were sealed and incubatedovernight at room temperature. Wells were then blocked with Superblock(Pierce) containing 2% goat serum (140-150 μl/well) for 1-2 hours atroom temperature. Plates were washed using an automated ELISA platewasher (twice with TBS 0.05% Tween20 using 200 μl/well). MIF samples andstandards were prepared in 0.5 ml or 1.5 ml eppendorf tubes by addingTween20 to culture supernatants to a final concentration of 0.2%. Celllysates were likewise diluted in TBS buffer with Tween20 at a finalconcentration of 0.2%. Standards were prepared similarly by dilutingpurified recombinant murine or human MIF in DMEM/1% FBS/0.2% Tween20.Samples and standards were applied to the plate (60 μl/well) and theplate sealed and incubated overnight at 4° C. with gentle shaking. Theplate was then washed five times with TBS/0.05% Tween20, and secondantibody (e.g. Rabbit 102 anti-murMIF serum, 1:220 in TBS/0.2%Tween20/2% goat serum) added at 60 μl/well. The plate was sealed andincubated 2 hours at room temperature with gentle shaking. All wellswere then washed five times with TBS/0.05% Tween20 and tertiaryantibody-enzyme conjugate (commercially available goat anti-rabbitIgG-alkaline phosphatase, diluted 1:4000 in TBS/0.2% Tween20/2% goatserum as recommended by the manufacturer, Boehringer Mannheim) was addedat 60 μl/well. The plate was covered, incubated for 35 minutes at roomtemperature, and then washed 5 times with TBS/0.05% Tween20. The assaywas then developed with p-nitrophenyl phosphate (pNPP) solution asrecommended by the manufacturer (5 mg Sigma 104 tablet in 5 ml APbuffer: 10 mM diethanolamine/0.5 mM MgCl₂, pH 9.5). Reaction product wasallowed to develop in the dark at room temperature, and read at 405 nmwithin 15-30 minutes. This assay gives range of sensitivity of about 100pg/ml-250 ng/ml. It should be noted that for the practice of this“sandwich” technique, various combinations of two or more MIF-specificantibodies may be used to capture and detect MIF in a sample. Theimmobilized antibody is not restricted to VIIG3 antibody, and the secondantibody is not limited to a rabbit antiserum.

The present invention is not to be limited in Scope by the exemplifiedembodiments which are intended as illustrations of single aspects of theinvention. Indeed, various modifications of the invention in addition tothose shown and described herein will become apparent to those skilledin the art from the foregoing description and accompanying drawings.Such modifications are intended to fall within the scope of the appendedclaims.

All publications cited herein are incorporated by reference in theirentirety.

1. A detection kit, comprising: (a) an antibody or antigen-bindingfragment or fusion protein thereof that specifically binds to humanmacrophage migration inhibitory factor (MIF) protein, wherein the humanMIF protein has a molecular weight of approximately 12.5 kDa asdetermined by SDS PAGE; (b) a detectable label, whereby the binding ofthe antibody or antigen-binding fragment or fusion protein to the humanMIF protein can be detected.
 2. The kit of claim 1, wherein (a) is theantibody.
 3. The kit of claim 1, wherein (a) is the antigen-bindingfragment.
 4. The kit of claim 1, wherein (a) is the fusion protein. 5.The kit of claim 1, wherein (a) is the antibody, and the antibody is amonoclonal antibody.
 6. The kit of claim 1, wherein the detectable labelcomprises one or more enzymes.
 7. The kit of claim 1, wherein thedetectable label comprises one or more fluorescent dyes.
 8. The kit ofclaim 1, wherein the detectable label comprises one or more coloredbeads.
 9. The kit of claim 1, wherein the detectable label comprises oneor more magnetic beads.
 10. The kit of claim 1, wherein the human MIFprotein exhibits MIF biological activity.
 11. A method of using thediagnostic kit of claim 1, comprising contacting a sample with (a) and(b).
 12. A method of making the diagnostic kit of claim 1, comprisingproviding (a) and (b) together to result in the diagnostic kit.
 13. Amethod for detecting human macrophage migration inhibitory factor (MIF)protein in a sample, comprising: (a) contacting the sample with the kitof claim 1; and (b) determining the presence or absence of human MIFprotein in the sample, wherein if the human MIF protein is detected,then the human MIF protein is present in the sample, and wherein if thehuman MIF protein is not detected, then the human MIF protein is notpresent in the sample.